An L-tryptophan-producing strain, its construction method and application

Abstract

The present invention provides an L-tryptophan-producing strain, a construction method and an application thereof. The strain lacks tnaA , serA、 trpR, trpL genes; the transcription of aroC , tktA genes is enhanced by using the tac promoter and is respectively integrated into the tnaA, trpR locus; the trpE gene in the genome is site-directed mutated to relieve feedback, and the feedback inhibition of tryptophan is relieved. The constructed strain has good tryptophan synthesis ability, stable performance and high acid production efficiency. In particular, by carrying the medium-copy plasmid pTtas, the finally obtained L-tryptophan-producing strain has a clear genetic background. The strain carrying the plasmid pTtas has no antibiotic screening label and does not require antibiotic pressure screening. It has good performance in producing L-tryptophan, high sugar-acid conversion rate, and can stably and efficiently produce or accumulate L-tryptophan by direct microbial fermentation method.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology production, in particular to an L-tryptophan production strain and a construction method and application thereof. Background Art

[0002] Tryptophan (L-trp) is a widely used aromatic amino acid. It not only participates in protein synthesis but also plays a crucial role in human immune regulation, serving as a precursor for serotonin and melatonin. In recent years, with the deepening understanding of L-tryptophan's biological activities and the expansion of its applications, global demand for L-tryptophan has increased dramatically, resulting in a shortage of supply. Therefore, increasing the industrial production capacity of L-tryptophan is of great practical significance.

[0003] The production method of L-tryptophan is mainly microbial fermentation, which uses cheap raw materials such as glucose as a carbon source and is achieved by microbial fermentation. In recent years, great progress has been made in constructing tryptophan strains using synthetic biology techniques, such as the tryptophan production strain constructed in patent CN111154706B, which can utilize Escherichia coli to efficiently synthesize tryptophan. Escherichia coli has a clear genetic background, mature gene editing technology, rapid growth, and the advantages of low nutritional requirements, making it an excellent starting strain in the microbial fermentation industry. However, due to the lengthy tryptophan synthesis pathway, involving the shikimic acid pathway, the chorismate pathway key enzymes are more, and the feedback mechanism is more complicated, resulting in limited output and conversion rate. To solve the above problems, it is necessary to release feedback inhibition from related enzyme genes and stably and efficiently express them. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an L-tryptophan producing strain.

[0005] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned L-tryptophan-producing strain.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned L-tryptophan producing strain.

[0007] In order to solve the above technical problems, the technical solution of the present invention is:

[0008] An L-tryptophan producing strain, named strain T5, is based on E.coli W3110 was used as the starting strain (chassis strain) and was obtained by gene editing using metabolic engineering: tnaA Gene, serA Gene (3-phosphoglycerate dehydrogenase gene) 、trpR Gene 、trpL gene; introduced aroCgene (chorismate synthase gene) and tktA Gene (transketolase gene), upregulated aroC Genes and tktA The transcription level of genes; trpE Genes undergo site-directed mutagenesis to resolve feedback. trpE The alanine at position 63 of the gene mutated to valine, and the cysteine ​​at position 465 mutated to tyrosine.

[0009] The above L-tryptophan production strain is enhanced by the tac promoter. aroC Gene, tktA Gene transcription and integration into tnaA Site 、trpR site, and the nucleotide sequence of the tac promoter is shown in the sequence listing SEQ ID NO.1.

[0010] Preferably, the L-tryptophan-producing strain also carries the pTtas plasmid and is named strain T6. The nucleotide sequence of the pTtas plasmid is shown in the sequence listing as SEQ ID NO.16.

[0011] Preferably, the L-tryptophan-producing strain (strain T6) is obtained by electroporating the pTtas plasmid into the competent state of the L-tryptophan-producing strain (strain T5).

[0012] The pTtas plasmid carries the replication origin, kanamycin resistance gene, tac promoter, endogenous promoter PserA, rrnB T1 terminator, and is used to efficiently express the feedback-resistant tryptophan operon from Escherichia coli. TrpE fbr DCBA Genes and aroG fbr Gene, artificial mini-operon serA fbr BC The E. coli tryptophan operon was expressed using the tac promoter. TrpE fbr DCBA Gene, aroG fbr gene, using the endogenous promoter PserA to drive the expression of serA fbr BC gene, the TrpE fbr The gene mutation sites are alanine at position 63 to valine (the TrpE fbrThe gene was subjected to site-directed mutagenesis, i.e., the base at position 188 was changed from C to T, resulting in the amino acid residue at position 63 being changed from alanine (A) to valine (V)), and the cysteine ​​at position 465 was changed to tyrosine (the base at position 1394 was changed from G to A, resulting in the amino acid residue at position 465 being changed from cysteine ​​(C) to tyrosine (Y)); aroG fbr The gene mutation site is serine at position 211 mutated to phenylalanine; serA fbr BC The gene is driven by the endogenous promoter PserA of Escherichia coli. serA fbr 、serB、serC The miniature artificial operon formed by the tandem expression of RBS sequences has a terminator of rrnB T1 terminator. Specifically, the miniature artificial operon serA fbr BC ,include serA fbr 、 serB 、 serC The genes are transcribed by the same E. coli endogenous promoter PserA and terminated by the same rrnB T1 terminator. The sequence of the operon genes is obtained by optimizing the gene connection mode and the position of the genes in the operon. serA fbr 、 serB 、 serC , rrnB T1 terminator, the PserA and serA fbr The genes are directly connected end to end. serC Directly linked end to end with the rrnB T1 terminator.

[0013] The above-mentioned microoperon serA fbr BC Using PserA to start, the series integration serA fbr 、 serB 、 serC , inserting the RBS sequence in the middle of the gene sequence to form a micro artificial operon that can express in series serA fbr The gene encoding 3-phosphoglycerate dehydrogenase, serB Encoded 3-phosphoserine phosphatase ,serC Encoded by 3-phosphoserine aminotransferase.

[0014] Feedback-decoupled tryptophan operon TrpE fbr DCBAgenetic trpE Site-directed mutagenesis is the same as above. aroG fbr The gene was subjected to site-directed mutation, that is, the base at position 632 was changed from G to A, resulting in the amino acid residue at position 211 being changed from serine (S) to phenylalanine (F); the above serA fbr The gene underwent site-directed mutagenesis, i.e., the bases at positions 1030, 1031, and 1032 were mutated from CAC to GCG, respectively, resulting in the change of the amino acid residue at position 344 from histidine (H) to alanine (A); the bases at positions 1036, 1037, and 1038 were mutated from AAC to GCG, respectively, resulting in the change of the amino acid residue at position 346 from asparagine (N) to alanine (A).

[0015] The nucleotide sequence of the endogenous promoter PserA is shown in the sequence listing SEQ ID NO.2; aroG fbr The gene (3-deoxy-7-phosphoheptanoate synthase mutant gene) is E.coli W3110 source aroG Gene site-directed mutagenesis, the nucleotide sequence of which is shown in the sequence table SEQ ID NO.10; tryptophan operon TrpE fbr DCBA The gene is derived from a strain of tryptophan genetically engineered bacteria, and its nucleotide sequence is shown in the sequence table SEQ ID NO.11; artificial mini-operon serA fbr BC The nucleotide sequence is shown in the sequence listing SEQ ID NO.12; serA fbr is E.coli W3110 source serA Gene site-directed mutagenesis, the nucleotide sequence of which is shown in the sequence listing SEQ ID NO.13; serB The nucleotide sequence is shown in the sequence listing SEQ ID NO.14; serC The nucleotide sequence is shown in the sequence listing as SEQ ID NO.15; the RBS nucleotide sequence is aggaa; and the rrnB T1 terminator nucleotide sequence is shown in the sequence listing as SEQ ID NO.17.

[0016] Preferably, the above-mentioned L-tryptophan producing strain, the starting strain E.coli W3110 is E.coli W3110 ATCC27325.

[0017] Preferably, the above-mentioned L-tryptophan producing strain, tnaAThe nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.3; serA The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.4; trpR The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.5; trpL The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.6.

[0018] Preferably, the above-mentioned L-tryptophan producing strain, aroC The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.7; tktA The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.8; after site-directed mutagenesis trpE Gene( trpE fbr , anthranilate synthase mutant gene) is shown in the sequence listing as SEQ ID NO.9.

[0019] The construction method of the above-mentioned L-tryptophan production strain is to E.coli W3110 is used as the starting strain (chassis strain), and the specific steps are as follows:

[0020] (1) Knockout of the gene encoding tryptophanase tnaA gene, and insert the gene encoding chorismate synthase at this site aroC gene, driven by the tac promoter;

[0021] (2) Knockout of the gene encoding the repressor protein trpR gene and insert the transketolase gene at this site tktA gene, driven by the tac promoter;

[0022] (3) Knockout of the leader peptide trpL gene, and at this locus trpE fbr Gene replacement trpLE Genes (including trpL and trpE ); trpE fbr Genes in the genome trpE The alanine at position 63 of the gene mutated to valine, and the cysteine ​​at position 465 mutated to tyrosine;

[0023] (4) Knockout of the gene encoding serine synthase serA Gene.

[0024] Preferably, in the above-mentioned method for constructing an L-tryptophan-producing strain, the pTtas plasmid is electroporated into the competent state of the genetically engineered bacteria obtained in step (4).

[0025] Application of the L-tryptophan producing strain in fermentation production of L-tryptophan.

[0026] Preferably, the application of the above-mentioned L-tryptophan production strain is carried out using a mechanically stirred fermenter, and the specific steps are as follows:

[0027] (1) Slant culture: Take the L-tryptophan-producing strain and inoculate it on the slant culture medium as the first generation slant culture, and culture it at 36-37℃ for 10-12 hours; take the colony of the first generation slant culture and inoculate it on the slant culture medium as the second generation slant culture, and culture it at 36-37℃ for 8-10 hours;

[0028] (2) Seed culture: The second generation slant was washed with sterile water and placed in the seed culture medium. The culture temperature was 36°C. The bioreactor was controlled to automatically add 25% ammonia solution to maintain the pH of the seed solution at 6.7±0.2 and the dissolved oxygen value at 45-55%. When the OD600nm of the seed solution reached 18, the next step of fermentation culture was carried out.

[0029] (3) Fermentation culture: The inoculation amount is 20%, the culture temperature is 37°C, and the bioreactor is controlled to automatically add 25% ammonia solution to maintain the seed liquid pH at 7.0±0.2 and the dissolved oxygen value at 45%-65%.

[0030] Preferably, in the application of the above-mentioned L-tryptophan production strain, the slant culture medium in step (1) is a universal LB solid culture medium.

[0031] Preferably, in the application of the above-mentioned L-tryptophan production strain, the seed culture medium in step (2) is: glucose 20-40 g / L, yeast extract powder 2-5 g / L, ammonium sulfate 1-5 g / L, potassium dihydrogen phosphate 1-5 g / L, anhydrous magnesium sulfate 0.5-2 g / L, ferrous sulfate heptahydrate 10-30 mg / L, manganese sulfate monohydrate 10-30 mg / L, V H 0.5-1.5 mg / L, V B1 0.5-1 mg / L, the rest is water.

[0032] Preferably, in the application of the above-mentioned L-tryptophan production strain, the fermentation medium used in step (3) is: glucose 20-40 g / L, yeast extract powder 2-5 g / L, ammonium sulfate 1-5 g / L, potassium dihydrogen phosphate 2-8 g / L, anhydrous magnesium sulfate 0.5-2 g / L, ferrous sulfate heptahydrate 25-60 mg / L, manganese sulfate monohydrate 20-40 mg / L, V H 0.1-0.5 mg / L, V B1 0.5-1 mg / L.

[0033] The above culture media can be prepared using standard methods.

[0034] Beneficial effects:

[0035] The above L-tryptophan producing strain is missing tnaA 、 serA、trpR、trpL gene, blocking the degradation pathway of tryptophan, inactivating the tryptophan repressor protein, and releasing the leader peptide that regulates the tryptophan operon; trpE The gene was subjected to double-site mutagenesis to relieve tryptophan feedback inhibition; the tac promoter was used to enhance aroC 、 tktA Gene transcription enhances the metabolic flux of the chorismate pathway and the supply of the precursor erythrose 4-phosphate; it has good tryptophan synthesis ability, stable performance and high acid production efficiency.

[0036] Furthermore, it carries the medium copy plasmid pTtas, which is highly efficient in expressing from Escherichia coli E.coli Feedback-decoupled tryptophan operon of W3110 TrpE fbr DCBA 、 aroG fbr Gene, artificial mini-operon serA fbr BC gene, which strengthens the tryptophan operon trpE fbr DCBA expression, strengthen serA fbr BC The expression strengthens aroG fbr expression; the key enzymes were expressed using an efficient and stable plasmid system, further strengthening the tryptophan metabolic flow. The resulting L-tryptophan production strain had a clear genetic background and achieved high-level tryptophan production.

[0037] This strain carries the plasmid pTtas, which has a non-antibiotic screening tag and does not require antibiotic pressure screening. It has good L-tryptophan production performance, a high sugar-acid conversion rate, and can stably and efficiently produce or accumulate L-tryptophan through microbial direct fermentation. Specifically:

[0038] The key enzymes of the tryptophan pathway are efficiently and stably expressed through the medium-copy plasmid pTtas. To prevent the plasmid from being lost during the growth of the strain, the tryptophan genetic engineering bacteria have knocked out the serA gene, resulting in serine deficiency. The plasmid pTtas expresses an artificial mini-operon. serA fbr BCThe gene, on the one hand, makes up for the serine synthesis pathway defect of the tryptophan genetic engineering bacteria and strengthens the serine pathway. Serine is a precursor for tryptophan synthesis and is conducive to tryptophan synthesis. On the other hand, the serine synthesis pathway of the genetically engineered bacteria depends on the expression of the plasmid pTtas. Loss of the plasmid in the strain causes serine deficiency and death of the strain. Therefore, the plasmid pTtas has a screening pressure on the tryptophan genetic engineering bacteria. The artificial mini-operon expressed by the plasmid serA fbr BC The gene acts as an anti-resistance selection label, which not only improves the plasmid stability during strain production, but also avoids the use of antibiotics. It has economic advantages and environmental safety advantages in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a map of plasmid pTtas.

[0040] Figure 2 This is a comparison chart of the yield of L-tryptophan producing strain T5 at different stages.

[0041] Figure 3 This is a comparison chart of the yield and conversion rate of L-tryptophan production strain T6 at different stages. DETAILED DESCRIPTION

[0042] 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.

[0043] The percentage sign "%" involved in the examples, unless otherwise specified, refers to mass percentage. The percentage of a solution refers to the number of grams of solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0044] The starting strain used in the embodiment is the wild type E.coli W3110 ATCC 27325, the corresponding promoter and gene are shown in the sequence listing. The primers used in the construction of the involved strains are shown in Table 1.

[0045] Table 1 Primers involved in strain construction

[0046] Primer name Sequence number Sequence tnaA-US SEQ ID NO.18 CGCGCCATCGCTCAGGTTGTACC tnaA-UA SEQ ID NO.19 CCGCTCACAATTCCACACATTATACGAGCCGATGATTAATTGTCAATACATAATCCTTCATTTATTTTAATTACAGTG tnaA-DS SEQ ID NO.20 GAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTAATACTACAGAGTGGC tnaA-DA SEQ ID NO.21 AGGCATAAGTGAGGATATAGAGAACG aroC-tac-s SEQ ID NO.22 TGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGTATTCATGATGGCTGGAAACACAATTGGACAACTC aroC-tac-a SEQ ID NO.23 CAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACCAGCGTGGAATATCAGTCTTCACATCGGC pGRB-tnaA-s SEQ ID NO.24 AGTCCTAGGTATAATACTAGTGTTCGGCCTGGCTGCGACTCGTTTTAGAGCTAGAA pGRB-tnaA SEQ ID NO.25 TTCTAGCTCTAAAACGAGTCGCAGCCAGGCCGAACACTAGTATTATACCTAGGACT trpR-US SEQ ID NO.26 GCCAGCGGCTTGATGCAGATTAT trpR-UA SEQ ID NO.27 GAAATTGTTATCCGCTCAATTCCACACATTATACGAGCCGATGATTAATTGTCAAAATATGTCGCCATTTGTTAGCG trpR-DS SEQ ID NO.28 GGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAATTTTTGTAGGCCTGATAAGACGTGGCGC trpR-DA SEQ ID NO.29 GTAGCCAATGCCCGCCGTTTA tktA-tac-s SEQ ID NO.30 TCCGGCTCGTATAATGTGTGGAATTTGTGAGCGGATAACAATTTCACAGGAAACAGACCATGACGGAAAACATTCATAAG tktA-tac-a SEQ ID NO.31 CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTCATTCCCACTCAATGGTA pGRB-trpR-s SEQ ID NO.32 AGTCCTAGGTATACTAGTCGTGAGTTAAAAAATGAACTGTTTTAGAGCTAGAA pGRB-trpR-a SEQ ID NO.33 TTCTAGCTCTAAAACAGTTCATTTTAACTCACGACTAGTATTATACCTAGGACT trpLE-1 SEQ ID NO.34 TTCTGTCTGCTGCGCGAGGAACT trpLE-2 SEQ ID NO.35 CCTAGAAGAAATCAACCAGCGCATCAGAAAGTCTCCTGTGCATTGTCGATACCCTTTTTACGTGAACT trpLE-3 SEQ ID NO.36 TGCGCTGGTTGATTTCTTCTAGGGTCATAGTAATCCAGCAACTATGGCTGACATTCTGCTGCTCGATA trpLE-4 SEQ ID NO.37 GTTCCAGCAGGCGAGCGCCCTG pGRB-LEs SEQ ID NO.38 AGTCCTAGGTATAATACTAGTCTCGAACTGCTAACCTGCGAGTTTTAGAGCTAGAA pGRB-LE-a SEQ ID NO.39 TTCTAGCTCTAAAACTCGCAGGTTAGCAGTTCGAGACTAGTATTATACCTAGGACT trpE-1 SEQ ID NO.40 TTCTGTCTGCTGCGCGAGGAACT trpE-2 SEQ ID NO.41 CCGCTCAATTCCACACATTATACGAGCCGATGATTAATTGTCAATGTCGATACCCTTTTTACGTG trpE-5 SEQ ID NO.42 TATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAATATGGCTGACATTCTGCTGCTCGATA trpE-6 SEQ ID NO.43 GTTCCAGCAGGCGAGCGCCCTG trpE-tac-S SEQ ID NO.44 AATGTGTGGAATTTGTGAGCGGATAACAATTTCACAGGAAACAGTATTCATGCAAACACAAAAACCG trpEtb1-A SEQ ID NO.45 CTGTAATGCGCAGCGCACT trpEtb1-S SEQ ID NO.46 AGTGCGCTGCGCATTACAGTTTTAGGTGACAC trpEtb2-A SEQ ID NO.47 AGGTGTCGAGATCGCCATGC trpEtb2-S SEQ ID NO.48 GCATGGCGATCTCGACACCTACATTGTGATCCGCTCGGC trpE-tac-A SEQ ID NO.49 CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTCAGAAAGTCTCCTGTGC pGRB-4s SEQ ID NO.50 AGTCCTAGGTATACTAGTGCGCTGGTTGATTTCTTCTAGTTTTAGAGCTAGAA pGRB-4-a SEQ ID NO.51 TTCTAGCTCTAAAACTAGAAGAAATCAACCAGCGCACTAGTATACCTAGGACT ΔserA-US SEQ ID NO.52 GCAAAGGTATCGCTGGAGAAAGAC ΔserA-UA SEQ ID NO.53 CAGAATACTTGATCAATTTACCCGCAATAAATACCGGGATCCCGCGCT ΔserA-DS SEQ ID NO.54 ACGCGGGATCCCGGTATTTATTGCGGGTAAATTGATCAAGTATTCTG ΔserA-DA SEQ ID NO.55 TTAGTACAGCAGACGGGCGC pGRB-serA-s SEQ ID NO.56 AGTCCTAGGTATATACTAGTTCCGTCCACCAAAAATATGAGTTTTAGAGCTAGAA in pGRB-serA SEQ ID NO.57 TTCTAGCTCTAAAACTCATATTTTTGGTGGACGGAACTAGTATTATACCTAGGACT trp-s SEQ ID NO.58 AACTTACATTAATTCGTTGCGCTTGACAATTAATCATCGGCTCGTATAATGTGT trp-A SEQ ID NO.59 CGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAATTTGTCCTACTCAGGAGAGC PET-line-S SEQ ID NO.60 GCTCTCCTGAGTGACAAATTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCG PET-line-A SEQ ID NO.61 ACACATTATACGAGCCGATGATTAATTGTCAAGCGCAACGCAATTAATGTAAGTT aroG-1 SEQ ID NO.62 CGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGTATTCATGATGAATTATCAGAACGACGATT aroG-2 SEQ ID NO.63 ACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACCCGCGACGCGCTTTTACTG aroGtb-A SEQ ID NO.64 CAGCGGCAGGTGAGTTTCTC aroGtb-S SEQ ID NO.65 TGAGTTTCTCAATATGATCACCCCAC PET2-line-S SEQ ID NO.66 TGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGGGATCTCGACGCTCTCCCCTTATG PET2-line-A SEQ ID NO.67 GTGTGAAATTGTTATCCGCTCAATTCCACACATTATACGAGCCGATGATTAATTGTCAAATTTGTCCTACTCAGGAGAGCG serA-1 SEQ ID NO.68 GCAGCCCAGTAGTAGGTTGAGGCCCGACGCAAAACGTTCATATTGCCGCAATA serA-344-2 SEQ ID NO.69 GGCGTCGTCTGATGCACATC serA-346-3 SEQ ID NO.70 GATGCACATCGCGGAAGCGCGTCCGGGC serA-4 SEQ ID NO.71 CGCACCAGGTAATGTTAGGCATTTCCTTTAGTACAGCAGACGGGCGCGAATGGTACC serB-1 SEQ ID NO.72 GGTACCATTCGCGCCCGTCTGCTGTACTAAAGGAAATGCCTAACATTACCTGGTGCG serB-2 SEQ ID NO.73 GAACTAAATTGAAGATTTGAGCCATTTCCTTTACTTCTGATTCAGGCTGCCTGAG serC-1 SEQ ID NO.74 CTCAGGCAGCCTGAATCAGAAGTAAAGGAAATGGCTCAAATCTTCAATTTTAGTTC serC-2 SEQ ID NO.75 GAAGGGATAAAAAAACGGGCAAGTCAGTGACCTGCCCGTTGATTTTCAGAGAAGGGGAATTAACCGTGACGGCGTTCGAACTC PET3-line-S SEQ ID NO.76 ACTGACTTGCCCGTTTTTTTATCCCTTCCCACACGTTGCCGCGTTGCCTGAAAGGAGGAACTATATCCGG PET3-line-A SEQ ID NO.77 TATTGCGGCAATATGAACGTTTGCGTCGGGCCTCAACCTACTACTGGGCTGC

[0047] In the following examples, L-tryptophan production strains were constructed by the following method:

[0048] (1) Knockout of the gene encoding tryptophanase tnaA gene, and insert the gene encoding chorismate synthase at this site aroC gene, and driven by the tac promoter;

[0049] (2) Knockout of the gene encoding the repressor protein trpR gene and insert the transketolase gene at this site tktA gene, and driven by the tac promoter;

[0050] (3) Knockout of the leader peptide trpL gene, and at this locus trpE fbr Gene replacement trpLE Genes (including trpL and trpE ); trpE fbr Genes in the genome trpE The alanine at position 63 of the gene mutated to valine, and the cysteine ​​at position 465 mutated to tyrosine;

[0051] (4) Knockout of the gene encoding serine synthase serA Gene;

[0052] (5) The constructed pTtas plasmid is electroporated into the genetically engineered bacteria obtained in step (4). The plasmid can efficiently express the feedback-decoding tryptophan operon from Escherichia coli. TrpE fbr DCBA , aroG fbr Gene, artificial mini-operon serA fbr BC Gene.

[0053] The gene editing methods used in the above-mentioned genetic manipulations are described in the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21). Unless otherwise noted, all technical terms used in this invention are explained in that article. "Knockout" as used in this invention refers to the inactivation of a target gene, and "introduction" refers to the insertion of a foreign gene into the genome of the engineered bacterium after ligation with a promoter and terminator.

[0054] Example 1

[0055] This example is intended to illustrate the specific steps for constructing the strain. In particular, if there is a method for manipulating the same type of gene in the examples, only one of them will be provided and annotated without further elaboration.

[0056] (1) Knockout tnaA gene and integrate aroC gene: E.coli The W3110 genome was used as a template, and tnaA-US / tnaA-UA, tnaA-DS / tnaA-DA, and aroC-tac-s / aroC-tac-a were used to amplify the upstream and downstream homology arms and target gene fragments respectively. Then, the upstream and downstream homology arms and target gene fragments were used as templates, and tnaA-US and tnaA-DA were used as primers to amplify the overlapping fragments by overlapping PCR. The gRNA fragments were annealed and ligated with the pGRB vector to obtain tnaA-pGRB. E. coli W3110 competent cells were electroporated, and the overlapping fragments and tnaA-pGRB were electroporated into competent cells together, and positive transformants were screened to obtain strain T1.

[0057] (2) Knockout trpR Gene and integration of tktA gene: The operation method is the same as (1), except that the primers used are trpR-US / trpR-UA, trpR-DS / trpR-DA, tktA-tac-s / tktA-tac-a, pGRB-trpR-s, pGRB-trpR-a, and the competent cell is T1, and the strain T2 is obtained.

[0058] (3) Knockout suffer gene and integrate Ptac- suffer fbr :by E. coli The W3110 genome was used as a template, and the DNA sequence of the exogenous cleavage site was segmented and added to the primers trpLE-2 and trpLE-3. The upstream and downstream homology arms were amplified by PCR using primers trpLE-1 and trpLE-2, trpLE-3 and trpLE-4, respectively. The recovered upstream and downstream homology arms were used as templates, and overlapping PCR was performed with primers trpLE-1 and trpLE-4 to obtain the knockout gene. suffer The complementing fragment containing the exogenous cleavage site was annealed using pGRB-LE-s and pGRB-LE-a as primers to obtain the gRNA fragment, which was then ligated with the pGRB vector to obtain pGRB-LE. Electroporation competent cells of strain T2 were prepared, and the target fragment and pGRB-LE were electroporated into the competent cells together. Positive transformants were screened to obtain strain T3.

[0059] Next, using Escherichia coli W3110 as a template, primers trpE-1 and trpE-2, trpE-5 and trpE-6 were used for PCR amplification to obtain upstream and downstream homology arms, and the mutant nucleotide sequence was designed on trpEtb1-A, trpEtb1-S, trpEtb2-A, trpEtb2-S, and there was a homologous sequence. The primers trpE-tac-S / trpEtb1-A, trpEtb1-S / trpEtb2-A, trpEtb2-S / trpE-tac-A were used for PCR amplification to obtain fragment 1, fragment 2 and fragment 3, respectively. Then, using fragment 1, fragment 2 and fragment 3 as templates, primers trpE-tac-S / trpE-tac-A were used for overlapping PCR to obtain the target fragment suffer fbr ; Then above the homology arm, suffer fbr The overlapping fragment P was obtained by overlapping PCR using primers trpE-1 / trpE-6. tac - suffer fbr ; Using pGRB-4-s and pGRB-4-a as primers, anneal the gRNA fragment and connect it with the pGRB vector to obtain pGRB-4; prepare strain T3 electroporation competent cells, electroporate the target fragment together with pGRB-4 into the competent cells, and screen to obtain positive transformants to obtain strain T4.

[0060] (4) Knockout serA Gene: E. coli The W3110 genome was used as a template, and the upstream and downstream homology arms were amplified by PCR using ΔserA-US, ΔserA-UA and ΔserA-DS, ΔserA-DA, respectively. Then, the upstream and downstream homology arms were used as templates and serA-US and serA-DA were used as primers to obtain overlapping fragments by overlapping PCR amplification. Using pGRB-serA-s and pGRB-serA-a as primers, the gRNA fragment was annealed to obtain the gRNA fragment, which was ligated with the pGRB vector to obtain serA-pGRB. The T4 strain was prepared for electroporation competent cells, and the overlapping fragments and serA-pGRB were electroporated into the competent cells together, and the positive transformants were screened to obtain the strain T5.

[0061] (5) Prepare electroporation competent cells of strain T5, electroporate the pTtas plasmid (the pTtas plasmid is obtained by the method described in Example 2) into T5 competent cells, and screen for positive transformants to obtain strain T6.

[0062] Example 2

[0063] This example is intended to illustrate the construction method of the pTtas plasmid in Example 1. The specific steps are as follows:

[0064] (1) Using the genome of strain T5 as template and trp-s and trp-A as primers, the primers contain tac promoter, and amplify suffer fbr DCBA Operator; using PET-line-S and PET-line-A as primers, amplify to obtain a linear vector, ligate the gene fragment with the linearized plasmid vector using a recombinase, transform into a DH5α competent cell, and screen for positive transformants for plasmid extraction to obtain a recombinant plasmid. Any recombinase can be used for the ligation method; in this example, ClonExpress® rapid cloning technology from Nanjing Novozymes Biotech Co., Ltd. was used to construct the overexpression plasmid.

[0065] (2) The same operation method as in (1) is used, except that the E. coli W3110 genome is used as a template to design primers aroG-1 and aroG-2. Then, the mutant nucleotide sequence is designed on aroGtb-A and aroGtb-S, and there are homologous sequences. PCR amplification is performed using primers aroG-1 / aroGtb-A and aroGtb-S / aroG-2 to obtain fragment 1 and fragment 2, respectively. Then, using fragment 1 and fragment 2 as templates, overlapping PCR is performed using primers aroG-1 / aroG-2 to obtain the target fragment P. tac -aroG fbr ;Use the recombinant plasmid in step 1 as a template, design PET2-line-S and PET2-line-A as primers to amplify and obtain a linear vector. Connect the gene fragment and the plasmid linearized vector through the recombinase, transform it into the DH5α competent medium, and screen the positive transformants for plasmid extraction to obtain the recombinant plasmid.

[0066] (3) Using the E. coli W3110 genome as a template, primers serA-1, serA-344-2, serA-346-3, and serA-4 were designed for serA The gene was subjected to site-directed mutagenesis to obtain P serA - serA fbr Fragment; designed primers are serB-1, serB-2, and the E. coli W3110 genome is used as a template to perform PCR amplification to obtain the serB fragment; designed primers are serC-1, serC-2, and the E. coli W3110 genome is used as a template to perform PCR amplification to obtain the serC fragment. RBS are designed on primers serA-4, serB-1, serB-2, and serC-1. serA fbr, serB, serC as templates, and primers serA-1 and serC-1 were used to amplify P serA - serA fbr BC. Using the recombinant plasmid from step 2 as a template, design primers PET3-line-S and PET3-line-A to amplify a linear vector. Connect the gene fragment to the linearized plasmid vector using a recombinase, transform it into a DH5α competent cell, and screen the positive transformants for plasmid extraction to obtain the recombinant plasmid pTtas. The nucleotide sequence of pTtas is shown in the sequence listing as SEQ ID NO.16, and the plasmid map is shown in Figure 1 .

[0067] Example 3

[0068] L-tryptophan was produced by fermentation in a 5 L fermentor using the strain T5 described in Example 1. The specific steps are as follows:

[0069] (1) Slant culture: Take the L-tryptophan-producing strain and inoculate it on the slant medium as the first generation slant culture, and culture it at 37℃ for 12 hours; 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 37℃ for 8 hours; the slant medium should be the universal LB solid medium;

[0070] (2) Seed culture: The second generation slant was washed with sterile water and placed in the seed culture medium. The culture temperature was 36°C. A 5L bioreactor was used to control the automatic flow of 25% ammonia solution to maintain the pH of the seed solution at 6.7, the dissolved oxygen value at 50%, and the OD of the seed solution at 1. 600nm When the temperature reaches 18, 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 3 g / L, potassium dihydrogen phosphate 4 g / L, anhydrous magnesium sulfate 1 g / L, ferrous sulfate heptahydrate 20 mg / L, manganese sulfate monohydrate 20 mg / L, V H 1 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 and the dissolved oxygen value was 50% by automatically adding 25% ammonia solution through the bioreactor control. The fermentation medium used was: glucose 20 g / L, yeast extract powder 5 g / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 6 g / L, anhydrous magnesium sulfate 1.4 g / L, ferrous sulfate heptahydrate 25 mg / L, manganese sulfate monohydrate 20 mg / L, V H 0.5 mg / L, V B11 mg / L, 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 0.1-1 g / L; the fermentation cycle is 48 hours.

[0072] like Figure 2 As shown in the figure, the maximum L-tryptophan yield of strain T5 in a 5 L fermenter reached 28.9 g / L.

[0073] Example 4

[0074] L-tryptophan was produced by fermentation in a 5 L fermentor using the strain T6 described in Example 1 (the strain T6 is a strain carrying the pTtas plasmid based on the strain T5), and the specific steps were the same as those in Example 3.

[0075] like Figure 3 As shown, strain T6 achieved a maximum L-tryptophan yield of 60 g / L in a 5 L fermentor, with a maximum sugar-to-acid conversion rate of 19% within 36 hours. This example employed a 5 L fermentor, which is relatively small. As is well known to those skilled in the art, this sugar-to-acid conversion rate would stabilize at 21%-22% upon scale-up, offering significant technical advantages.

[0076] 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 considered to be within the scope of protection of the present invention.

Claims

1. An L-tryptophan producing strain, characterized in that: So E. coli W3110 was used as the starting strain and gene editing was performed using metabolic engineering: tnA Gene, serA Gene trpR Gene ,trpL gene; introduced aroC Genes and TKMv Gene; trpE Genes are subjected to site-directed mutagenesis to resolve feedback. trpE The alanine at position 63 of the gene is mutated to valine, and the cysteine ​​at position 465 is mutated to tyrosine; and the pTtas plasmid is carried at the same time, and the nucleotide sequence of the pTtas plasmid is shown in the sequence table SEQ ID NO.16; aroC The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.7; TKMv The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO.8; after site-directed mutagenesis trpE The nucleotide sequence of the gene is shown in the sequence listing SEQ ID NO.

9.

2. The L-tryptophan producing strain according to claim 1, characterized in that: The starting strain E. coli W3110 is E. coli W3110 ATCC 27325.

3. The method for constructing the L-tryptophan producing strain according to claim 1, characterized in that: by E. coli W3110 was used as the starting strain, and the specific steps were as follows: (1) Knockout of the gene encoding tryptophanase tnA gene, and insert the gene encoding chorismate synthase at this site aroC gene, driven by the tac promoter; (2) Knockout of the gene encoding the repressor protein trpR gene, and insert the transketolase gene at this site TKMv gene, driven by the tac promoter; (3) Knockout of leader peptide tL gene, and at this locus trpE fbr Gene replacement trpL gene; trpE fbr Genes in the genome trpE The alanine at position 63 of the gene mutated to valine, and the cysteine ​​at position 465 mutated to tyrosine; (4) Knockout of the gene encoding serine synthase serA Gene; (5) The pTtas plasmid is electroporated into the competent cell of the genetically engineered bacteria obtained in step (4), wherein the nucleotide sequence of the pTtas plasmid is shown in the sequence listing as SEQ ID NO.

16.

4. Use of the L-tryptophan producing strain according to claim 1 in fermentation production of L-tryptophan.

5. The use according to claim 4, characterized in that: Fermentation is carried out using a fermentation tank. The specific steps are as follows: (1) Slant culture: Take the L-tryptophan producing strain and inoculate it on the slant medium as the first generation slant culture, and culture it at 36-37℃ for 10-12h; take the colony of the first generation slant culture and inoculate it on the slant medium as the second generation slant culture, and culture it at 36-37℃ for 8-10h; (2) Seed culture: The second-generation slant was washed with sterile water and placed in the seed culture medium. The culture temperature was 36°C. The bioreactor was controlled to automatically add ammonia solution to maintain the pH of the seed solution at 6.7±0.2 and the dissolved oxygen value at 45-55%. When the OD600nm of the seed solution reached 18, the next step of fermentation culture was carried out. (3) Fermentation culture: The inoculation amount is 20%, the culture temperature is 37°C, and the bioreactor is controlled to automatically add ammonia solution to maintain the pH of the seed liquid at 7.0±0.2 and the dissolved oxygen value at 45%-65%.

6. The use according to claim 5, characterized in that: The seed culture medium in step (2) is: 20-40 g / L glucose, 2-5 g / L yeast extract powder, 1-5 g / L ammonium sulfate, 1-5 g / L potassium dihydrogen phosphate, 0.5-2 g / L anhydrous magnesium sulfate, 10-30 mg / L ferrous sulfate heptahydrate, 10-30 mg / L manganese sulfate monohydrate, V H 0.5-1.5 mg / L, V B1 0.5-1 mg / L, the rest is water.

7. The use according to claim 5, characterized in that: The fermentation medium used in step (3) is: 20-40 g / L glucose, 2-5 g / L yeast extract powder, 1-5 g / L ammonium sulfate, 2-8 g / L potassium dihydrogen phosphate, 0.5-2 g / L anhydrous magnesium sulfate, 25-60 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.

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