CN118086167B - Genetically engineered bacterium for producing L-tryptophan and construction method and application thereof - Google Patents
Genetically engineered bacterium for producing L-tryptophan and construction method and application thereofInfo
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- CN118086167B CN118086167B CN202410519412.XA CN202410519412A CN118086167B CN 118086167 B CN118086167 B CN 118086167B CN 202410519412 A CN202410519412 A CN 202410519412A CN 118086167 B CN118086167 B CN 118086167B
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- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims abstract description 27
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Abstract
The invention provides a genetic engineering bacterium for producing L-tryptophan and a construction method and application thereof, wherein the genetic engineering bacterium lacks tnaA, tyrR, trpR genes, up-regulates the transcription level of genes trpE fbr、aroGfbr、serAfbr and talB, aroB, aroD, aroE, aroK, aroA, aroC, yddG, strengthens the expression of tryptophan operon, and heterologously introduces a phosphoketolase gene bbxfpk derived from bifidobacterium breve and a glutamine synthetase gene glnA fbr derived from bacillus subtilis; the L-tryptophan production strain obtained finally has clear genetic background, good L-tryptophan production capacity, stable performance and high sugar-acid conversion rate, and can stably and efficiently produce or accumulate L-tryptophan by a microorganism direct fermentation method.
Description
Technical Field
The invention relates to the field of biotechnology production, in particular to a genetically engineered bacterium for producing L-tryptophan as well as a construction method and application thereof.
Background
Tryptophan (L-tryptophan, L-trp), the chemical name of which is alpha-amino-beta-indolepropionic acid, is an aromatic amino acid with wide application, not only participates in protein synthesis, but also plays a vital role in the immune regulation of human body, and is a synthesis precursor of serotonin and melatonin. In recent years, as people continuously and deeply know the bioactivity of L-tryptophan and the application field thereof is continuously expanded, the demand of the global market for L-tryptophan is rapidly increased, and the supply of L-tryptophan is not required, so that the improvement of the industrial productivity of L-tryptophan has very practical significance.
The production mode of L-tryptophan is mainly a microbial fermentation method, and the microbial fermentation method is realized by taking cheap raw materials such as glucose and the like as carbon sources and adopting a microbial fermentation mode. In the early research process, researchers only use traditional chemical or physical mutagenesis modes to cultivate the L-tryptophan production strain, and the production strain obtained by the mode has poor stability and low acid production performance. The directional transformation by the genetic engineering means is hopeful to make up for the shortages of mutation breeding modes.
The escherichia coli has the advantages of clear genetic background, mature gene editing technology, rapid growth and low nutrition requirement, and is an excellent original strain in the microbial fermentation industry. At present, the problem of low yield and sugar acid conversion rate of the L-tryptophan produced by the engineering bacteria of the escherichia coli mainly exists, the sugar acid conversion rate of the main flow tryptophan production strain in the domestic market is only 17% -18%, the raw material cost is high, and the productivity is severely restricted, so that the improvement of the sugar acid conversion rate and the yield of the L-tryptophan production strain is a technical problem which needs to be solved at present.
Disclosure of Invention
The invention aims to provide a genetically engineered bacterium for producing L-tryptophan.
The invention aims to provide a construction method of the genetically engineered bacterium for producing L-tryptophan.
The invention aims to provide an application of the genetically engineered bacterium for producing L-tryptophan.
In order to solve the technical problems, the technical scheme of the invention is as follows:
A genetically engineered bacterium for producing L-tryptophan is a strain YTP17, lacks tnaA, tyrR, trpR genes, up-regulates the transcription level of trpE fbr、aroGfbr、serAfbr and talB, aroB, aroD, aroE, aroK, aroA, aroC, yddG genes, strengthens the expression of TrpE fbr D, trpDC, trpBA in each gene cluster in tryptophan operon TrpE fbr DCBA, and introduces a phosphoketolase gene bbxfpk derived from bifidobacterium breve and a glnA fbr gene derived from bacillus subtilis in a heterologous way.
Preferably, the genetically engineered strain for producing L-tryptophan uses E.coli W3110 as a starting strain (chassis strain) and performs gene editing by using metabolic engineering means:
(1) Knocking out the tnaA gene encoding tryptophan enzyme;
(2) Knocking out tyrR and trpR genes encoding the repressor proteins TyrR and TrpR;
(3) The original trpLE gene is replaced by an anthranilate synthase mutant trpE fbr gene with feedback inhibition released, and the trc promoter is used for starting;
(4) The 3-deoxy-7-phosphoheptanoic acid synthase mutant gene aroG fbr for enzymolysis and feedback inhibition removal is integrated in series at a pseudogene locus yjiV and is started by a trc promoter;
(5) The talB gene is respectively integrated at the pseudogene locus ilvG and yeeL locus; and all are started by trc promoter;
(6) At pseudolocus yghE, a phosphoketolase gene bbxfpk encoding a bifidobacterium source is integrated and is used in combination with a trc promoter;
(7) The aroB, aroD, aroE genes are integrated in tandem at the yjgX locus of the genome to form a mini-operon aroBDE, which is then promoted by the trc promoter;
(8) The aroK, aroA and aroC genes are integrated in series at the ylbE locus of the genome to form a micromanipulator aroKAC, and the micromanipulator is started by a trc promoter;
(9) The tryptophan operon TrpE fbr DCBA is split into three micromanipulators TrpE fbr D, trpDC and TrpBA, and the three micromanipulators TrpE fbr D, trpDC and TrpBA are respectively integrated into the yjiV, yjiT and yjiP pseudogene loci and all are started by using the BBa_j23101 promoter;
(10) The phosphoglycerate dehydrogenase mutant gene serA fbr is integrated at a pseudogene locus ycdN and is started by a trc promoter;
(11) Integrating a bacillus subtilis-derived glutamine synthetase mutant glnA fbr gene at a genome mbhA site, and using a trc promoter to start;
(12) The yddG gene is integrated at the rph locus and yjiK locus of the genome; and all were started with trc promoter.
Preferably, in the above genetically engineered bacterium for producing L-tryptophan, the trpE fbr gene is subjected to site-directed mutagenesis, i.e., the bases at positions 118 and 119 are both C to T, resulting in the change of the 40 th amino acid residue from serine (S) to phenylalanine (F); site-directed mutagenesis of the aroG fbr gene was performed, i.e., the base at position 436 was changed from G to A, resulting in the change of amino acid residue 146 from aspartic acid (D) to asparagine (N); site-directed mutagenesis was performed on the serA fbr gene, i.e., the bases at positions 1030, 1031, 1032 were mutated from CAC to GCG, respectively, resulting in the change of amino acid residue 344 from histidine (H) to alanine (A); mutation of the base at positions 1036, 1037, 1038 from AAC to GCG, respectively, results in the change of the amino acid residue at position 346 from asparagine (N) to alanine (a); the glnA fbr gene is subjected to site-directed mutagenesis, namely, the base at 475 th and 477 th are respectively mutated from TA to AC, so that 159 th amino acid residue is changed from leucine (L) to isoleucine (I); at positions 911 and 912, the base was mutated from AA to CG, respectively, resulting in the change of amino acid residue 304 from glutamic acid (E) to alanine (A).
Preferably, the above genetically engineered strain for producing L-tryptophan, wherein the mini-operon aroBDE comprises aroB, aroD, aroE gene, wherein the transcription is started by the same trc promoter and the RBS ribosome binding site controlled operon, and the transcription is stopped by the same rrnB T1 terminator controlled operon, and the mini-operon is obtained by optimizing the gene connection mode and the existing position of the gene in the operon, specifically:
(1) The sequence of the operon gene is trc promoter, RBS, aroB, aroD, aroE, rrnB T1 terminator;
(2) the trc promoter and RBS ribose binding site are directly connected with aroB gene end to end;
(3) aroE and rrnB T1 terminators are directly joined end to end.
The micromanipulator aroBDE is started by utilizing a trc promoter at the genome level, aroB, aroD, aroE is integrated in series, and an RBS sequence is inserted in the middle of the gene sequence, so that a micromanipulator is formed, and can be used for expressing the aroB gene coded 3-dehydroquinolinate synthase, aroD coded 3-dehydroquinolinate dehydratase and aroE coded shikimate dehydrogenase in series.
Preferably, the above genetically engineered strain for producing L-tryptophan, wherein the mini-operon aroKAC comprises aroK, aroA, aroC gene, wherein the transcription is started by the same trc promoter and the RBS ribosome binding site controlled operon, and the transcription is stopped by the same rrnB T1 terminator controlled operon, and the mini-operon is obtained by optimizing the gene connection mode and the existing position of the gene in the operon, specifically:
(1) The sequence of the operon gene is trc promoter, RBS, aroK, aroA, aroC, rrnB T1 terminator;
(2) the trc promoter and RBS ribose binding site are directly connected with aroK gene end to end;
(3) aroC and rrnB T1 terminators are directly joined end to end.
The micromanipulator aroKAC is started by utilizing a trc promoter at the genome level, aroK, aroA, aroC is integrated in series, and an RBS sequence is inserted in the middle of the gene sequence, so that a micromanipulator is formed, and shikimate kinase coded by aroK gene, 3-phosphoshikimate-1-carboxyvinyl transferase coded by aroA and chorismate synthase coded by aroC can be expressed in series.
Preferably, the engineering bacteria for producing L-tryptophan are characterized in that the modified tryptophan operon TrpE fbr DCBA is split into three mini-artificial operons TrpE fbr D, trpDC and TrpBA, and the three mini-artificial operons are respectively integrated into yjiV, yjiT, yjiP pseudogene loci, and the BBa_j23101 promoter is used for promoting transcription.
The artificial micromanipulator TrpE fbr D is obtained by splitting the modified tryptophan operon TrpE fbr DCBA, the bba_j23101 promoter and the RBS ribosome binding site control operon start transcription, and the same rrnB T1 terminator control operon stops transcription, specifically:
(1) The sequence of the operon gene is BBa_j23101 promoter, RBS, trpE fbr D and rrnB T1 terminator respectively;
(2) The BBa_j23101 promoter and RBS ribose binding site are respectively and directly connected with the TrpE fbr gene end to end;
(3) The TrpE fbr D gene uses a gene sequence shown in a sequence table SEQ ID NO. 21;
(4) The TrpD is directly connected with the rrnB T1 terminator end to end.
The operon TrpDC is obtained by splitting the modified tryptophan operon TrpE fbr DCBA, and the bba_j23101 promoter and RBS ribosome binding site control operon start transcription, and the same rrnB T1 terminator control operon stop transcription, specifically:
(1) The sequence of the operon gene is bba_j23101 promoter, RBS, trpDC, rrnB T1 terminator;
(2) The BBa_j23101 promoter and RBS ribose binding site are respectively connected with the head and the tail of the TrpD gene directly;
(3) TrpDC gene uses gene sequence shown in sequence table SEQ ID NO. 22;
(4) TrpC are respectively connected with the rrnB T1 terminator end to end directly.
The operon TrpBA is obtained by splitting the modified tryptophan operon TrpE fbr DCBA, and the bba_j23101 promoter and RBS ribosome binding site control operon start transcription, and the same rrnB T1 terminator control operon stop transcription, specifically:
(1) The sequence of the operon gene is BBa_j23101 promoter, RBS, trpBA and rrnB T1 terminator respectively;
(2) The BBa_j23101 promoter and RBS ribose binding site are respectively connected with the TrpB genes end to end directly;
(3) The TrpBA gene uses a gene sequence shown in a sequence table SEQ ID NO. 23;
(4) TrpA are respectively connected with the rrnB T1 terminator end to end directly.
Preferably, the above genetically engineered strain for producing L-tryptophan is E.coli W3110 ATCC 27325.
Preferably, the nucleotide sequence of the trc promoter of the genetically engineered bacterium for producing L-tryptophan is shown as a sequence table SEQ ID NO. 1; the nucleotide sequence of the BBa_j23101 promoter is shown as a sequence table SEQ ID NO. 2; the nucleotide sequence of RBS is shown in sequence table SEQ ID NO. 3; the nucleotide sequence of the rrnB T1 terminator is shown in a sequence table SEQ ID NO. 4; the nucleotide sequence of the tnaA gene is shown in a sequence table SEQ ID NO. 5; the nucleotide sequence of the tyrR gene is shown in a sequence table SEQ ID NO. 6; the nucleotide sequence of the trpR gene is shown in a sequence table SEQ ID NO. 7; the anthranilate synthase mutant gene trpE fbr is a trpE gene site-directed mutation derived from E.coli W3110, and the nucleotide sequence of the gene trpE is shown as a sequence table SEQ ID NO. 8; the aroG fbr of the 3-deoxy-7-phosphoheptanoic acid synthase mutant gene is obtained by site-directed mutagenesis of an aroG gene derived from E.coli W3110, and the nucleotide sequence of the aroG gene is shown as SEQ ID NO.9 of a sequence table; the talB gene is derived from E.coli W3110, and the nucleotide sequence of the talB gene is shown in a sequence table SEQ ID NO. 10; the bbxfpk gene is derived from bifidobacterium breve (Bifidobacterium brevis), and the nucleotide sequence of the bbxfpk gene is shown in a sequence table SEQ ID NO. 11; the nucleotide sequence of aroB gene is shown in sequence table SEQ ID NO. 12; the nucleotide sequence of aroD gene is shown in sequence table SEQ ID NO. 13; the nucleotide sequence of aroE gene is shown in sequence table SEQ ID NO. 14; The nucleotide sequence of the mini-operon aroBDE is shown in a sequence table SEQ ID NO. 15; the nucleotide sequence of aroK gene is shown in sequence table SEQ ID NO. 16; the nucleotide sequence of aroA gene is shown in sequence table SEQ ID NO. 17; the nucleotide sequence of aroC gene is shown in sequence table SEQ ID NO. 18; the nucleotide sequence of the mini-operon aroKAC is shown in a sequence table SEQ ID NO. 19; the nucleotide sequence of the modified tryptophan operon TrpE fbr DCBA is shown in a sequence table SEQ ID NO. 20; The nucleotide sequence of the mini-operon TrpE fbr D is shown in a sequence table SEQ ID NO. 21; the nucleotide sequence of the mini-operon TrpDC is shown in a sequence table SEQ ID NO. 22; the nucleotide sequence of the mini-operon TrpBA is shown in a sequence table SEQ ID NO. 23; the phosphoglycerate dehydrogenase mutant gene serA fbr is a site-directed mutation of a serA gene derived from E.coli W3110, and the nucleotide sequence of the mutant is shown as SEQ ID No.24 of a sequence table; The glutamine synthetase mutant glnA fbr gene is derived from bacillus subtilis, and the nucleotide sequence of the glutamine synthetase mutant glnA fbr gene is shown in a sequence table SEQ ID NO. 25; the yddG gene is derived from E.coli W3110, and the nucleotide sequence of the yddG gene is shown in a sequence table SEQ ID NO. 26.
The construction method of the genetically engineered bacterium for producing L-tryptophan comprises the following specific steps:
(1) Constructing chassis strains: taking E.coli W3110 as an original strain, and knocking out an L-tryptophan degradation pathway gene tnaA; knocking out repressor regulatory protein gene tyrR and trpR; introducing a 3-deoxy-7-phosphoheptanoic acid synthase gene aroG fbr after the solution feedback, and inserting a trpLE gene in a tryptophan operon as a pseudogene locus into a trpE fbr gene;
(2) Strengthening the supply of the aromatic precursor erythrose 4-phosphate: integrating the transaldolase gene talB in a double copy form in the genome; the phosphoketolase gene bbxfk derived from bifidobacterium breve is introduced in the heterologous way, and the genes are all transcribed by using trc promoter;
(3) Strengthening shikimic acid and branching acid pathway carbon flux: shikimic acid and key enzymes of the branched acid pathway are expressed in series to construct artificial operons aroBDE and aroKAC, and transcription of the artificial operon is enhanced through a trc promoter;
(4) Optimizing tryptophan operon expression: the tryptophan operon TrpE fbr DCBA was split into three artificial operons TrpE fbr D, trpDC, trpBA and integrated into pseudogene sites yjiV, yjiT, yjiP, all using trc promoters to enhance transcription;
(5) Improving serine and glutamine supply in tryptophan synthesis pathway: serine is one of direct precursors for synthesizing tryptophan, and glutamine provides amino for synthesizing tryptophan, so that a glutamine synthesis enzymolysis feedback gene glnA fbr from bacillus subtilis is introduced into a genome over-expressed D-3-phosphoglycerate dehydrogenation enzymolysis feedback gene serA fbr in a heterologous manner, and the genes are all transcribed by using a trc promoter;
(6) Enhancing L-tryptophan output ability: the gene yddG of the aromatic export protein is overexpressed in the genome in double copy form, and both genes are transcribed using the trc promoter.
The genetically engineered bacterium for producing L-tryptophan is applied to fermentation production of L-tryptophan.
Preferably, the application of the genetically engineered bacteria for producing L-tryptophan uses a mechanically stirred fermenter for fermentation, and the specific steps are as follows:
(1) Slant culture: inoculating L-tryptophan producing strain on slant culture medium as first generation slant culture, and culturing at 36-37deg.C for 12-16 hr; the slant culture medium is a universal LB solid culture medium; inoculating the colony of the first generation slant culture to a new slant culture medium as a second generation slant culture, and culturing at 36-37 ℃ for 10-12h;
(2) Seed culture: washing the second generation inclined plane with sterile water to a seed culture medium, wherein the culture temperature is 36 ℃, controlling the automatic feeding of 25% ammonia water solution through a bioreactor to maintain the pH value of seed liquid at 7.0+/-0.2, and the dissolved oxygen value of the seed liquid is 30-50%, wherein the OD 600nm of the seed liquid reaches 15, and then carrying out the next fermentation culture;
(3) Fermentation culture: the inoculation amount is 20%, the culture temperature is 37 ℃, the pH of the seed solution is maintained at 7.0+/-0.2 by controlling automatic feeding of 25% ammonia water solution through a bioreactor, and the dissolved oxygen value is 30% -50%.
Preferably, the application of the genetically engineered bacterium for producing L-tryptophan, wherein the seed culture medium in the step (2) is: 20-40 g/L of glucose, 2-5 g/L of yeast extract powder, 1-5 g/L of ammonium sulfate, 1-5 g/L of monopotassium phosphate, 0.5-2 g/L of anhydrous magnesium sulfate, 10-30 mg/L of ferrous sulfate heptahydrate, 10-30 mg/L of manganese sulfate monohydrate, 0.5-1 mg/L of V H0.1-0.5 mg/L,VB1 and the balance of water.
Preferably, the fermentation medium in the step (3) is: 20-40 g/L of glucose, 2-5 g/L of yeast extract powder, 1-5 g/L of ammonium sulfate, 1-5 g/L of monopotassium phosphate, 0.5-2 g/L of anhydrous magnesium sulfate, 30-60 mg/L of ferrous sulfate heptahydrate, 20-40 mg/L of manganese sulfate monohydrate and 0.5-1 mg/L of V H0.1-0.5 mg/L,VB1 .
The above culture medium can be prepared by standard method.
The beneficial effects are that:
The genetically engineered bacterium for producing the L-tryptophan strengthens the supply of aromatic amino acid synthesis raw materials of 4-phosphoric acid erythrose and phosphoenolpyruvic acid by analyzing and reconstructing metabolic flows related to tryptophan in escherichia coli and introducing phosphoketolase from bifidobacterium breve in a heterologous way; the heterologous introduction of bacillus subtilis-derived glutamine synthetase enhances the supply of amino donor-glutamine in the tryptophan synthesis pathway; in addition, the enhancement of shikimic acid pathway and chorismate pathway is realized by constructing the artificial operon enhancement gene cluster tandem expression, the synthesis pathway of serine which is a precursor of tryptophan synthesis and an L-tryptophan output system are optimized, and the finally obtained L-tryptophan production strain has clear genetic background, good L-tryptophan production capacity, stable performance and high sugar acid conversion rate, can stably and efficiently produce or accumulate L-tryptophan by a microorganism direct fermentation method, and can achieve the final yield of 52.3g/L by fermentation in a 5L fermentation tank for 40h, wherein the conversion rate reaches 20 percent, and has good application value.
Drawings
FIG. 1 is a fermentation process curve of genetically engineered bacterium YTP17 for producing L-tryptophan.
Detailed Description
In order to enable those skilled in the art to better understand the technical scheme of the present invention, the technical scheme of the present invention will be further described in detail below with reference to the specific embodiments.
The percentage "%" referred to in the examples is the mass percentage, the percentage of the solution is the gram of the solute contained in 100mL, and the percentage between the liquids is the volume ratio of the solution at 25 ℃.
The starting strain used in the examples was wild E.coli W3110 ATCC 27325, and the corresponding promoters and genes etc. are shown in the sequence listing. The primers used in the construction of the related strains are shown in Table 1.
TABLE 1 primers involved in the construction of strains
| Primer name | Sequence number | Primer sequence (5 '-3') |
| tnaA-US | SEQ ID NO.27 | ATTGATGGTCTTGAACAATTGGC |
| tnaA-UA | SEQ ID NO.28 | GAAGCTGTCGTCTTTCATGCACATTTTACTGGCTCAATAACACGAATG |
| tnaA-DS | SEQ ID NO.29 | CATTCGTGTTATTGAGCCAGTAAAATGTGCATGAAAGACGACAGCTTC |
| tnaA-DA | SEQ ID NO.30 | TCATGATGCCACCTTTAGAGGAA |
| pGRB-tnaA-s | SEQ ID NO.31 | AGTCCTAGGTATAATACTAGTGTTCGGCCTGGCTGCGACTCGTTTTAGAGCTAGAA |
| pGRB-tnaA-a | SEQ ID NO.32 | TTCTAGCTCTAAAACGAGTCGCAGCCAGGCCGAACACTAGTATTATACCTAGGACT |
| tyrR-US | SEQ ID NO.33 | TACAGCCCGAAAAGGCCGGAA |
| tyrR-UA | SEQ ID NO.34 | GGATACGGGATAACTGAGTTTGCCTTTTTCCCGATCGCCCAACAG |
| tyrR-DS | SEQ ID NO.35 | CTGTTGGGCGATCGGGAAAAAGGCAAACTCAGTTATCCCGTATCC |
| tyrR-DA | SEQ ID NO.36 | TGAAAACGTTGCCATACTCAAAAGGC |
| pGRB-tyrR-s | SEQ ID NO.37 | AGTCCTAGGTATAATACTAGTTGCCATAAATCCATCGAACAGTTTTAGAGCTAGAA |
| pGRB-tyrR-a | SEQ ID NO.38 | TTCTAGCTCTAAAACTGTTCGATGGATTTATGGCAACTAGTATTATACCTAGGACT |
| trpR-US | SEQ ID NO.39 | TGGCGTTAAACAGCGTCACGACT |
| trpR-UA | SEQ ID NO.40 | TTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATCATGATGAACCTCGGCTAACAAAGTG |
| trpR-DS | SEQ ID NO.41 | GGAGGCGGCAGTCACTATATGAATGACTTTATGGATTAACGGTGACTGGAT |
| trpR-DA | SEQ ID NO.42 | TCATCGCCGTCACTTCGGTTG |
| pGRB-trpR-s | SEQ ID NO.43 | CCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAAGAGACGATTAGTACTGGAA |
| pGRB-trpR-a | SEQ ID NO.44 | CGTGGAACCGCTTAGCAATGGCAAGCCCTTCTTTGTCCTTATCAGCTACTGTTAAAAGCAC |
| trpLE-1 | SEQ ID NO.45 | TTCTGTCTGCTGCGCGAGGAACT |
| trpLE-2 | SEQ ID NO.46 | CCTAGAAGAAATCAACCAGCGCATCAGAAAGTCTCCTGTGCATTGTCGATACCCTTTTTACGTGAACT |
| trpLE-3 | SEQ ID NO.47 | TGCGCTGGTTGATTTCTTCTAGGGTCATAGTAATCCAGCAACTATGGCTGACATTCTGCTGCTCGATA |
| trpLE-4 | SEQ ID NO.48 | GTTCCAGCAGGCGAGCGCCCTG |
| pGRB-LE-s | SEQ ID NO.49 | AGTCCTAGGTATAATACTAGTCTCGAACTGCTAACCTGCGAGTTTTAGAGCTAGAA |
| pGRB-LE-a | SEQ ID NO.50 | TTCTAGCTCTAAAACTCGCAGGTTAGCAGTTCGAGACTAGTATTATACCTAGGACT |
| trpE-1 | SEQ ID NO.51 | GGGTGACTTATGCGAAGCTCGAGCTTTAATCTCTGCCCCGTCG |
| trpE-2 | SEQ ID NO.52 | TACGCTTAATGCAGCAACAGTGGTT |
| trpE-3 | SEQ ID NO.53 | AGTTCACGTAAAAAGGGTATCGACATTGACAATTAATCATCCGGCTCGTA |
| trpE-4 | SEQ ID NO.54 | TATCGAGCAGCAGAATGTCAGCCATTCAGAAAGTCTCCTGTGCATGATGC |
| trpE-5 | SEQ ID NO.55 | GCATCATGCACAGGAGACTTTCTGAATGGCTGACATTCTGCTGCTCGATA |
| trpE-6 | SEQ ID NO.56 | TTCTAGCTCTAAAACTTTTGGCATTGCGTGTACGTACTAGTATTATACCTAGGACT |
| trpEtb-A | SEQ ID NO.57 | TGTTTTTTGATCTCACCCGGTAAAGTCG |
| trpEtb-S | SEQ ID NO.58 | CTAGCACAGTACCTAGGACTGAGCTAGCTGTCAATGTTGCCATTTCCAGCCCACCA |
| pGRB-1-s | SEQ ID NO.59 | AGTCCTAGGTATAATACTAGTTGCGCTGGTTGATTTCTTCTGTTTTAGAGCTAGAA |
| pGRB-1-a | SEQ ID NO.60 | TTCTAGCTCTAAAACAGAAGAAATCAACCAGCGCAACTAGTATTATACCTAGGAT |
| yjiV-US | SEQ ID NO.61 | TGTGACTGTGGAAGCCCTGTAT |
| yjiV-UA | SEQ ID NO.62 | TTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAATTCGGGCTGTCCCTTGTC |
| yjiV-DS | SEQ ID NO.63 | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGTGGCACCTGAATGACGA |
| yjiV-DA | SEQ ID NO.64 | TGGCGACATTCCCTTCCTT |
| aroG-3 | SEQ ID NO.65 | ATGAATTATCAGAACGACGATTTACGCA |
| aroG-4 | SEQ ID NO.66 | TAAAAGCGCGTCGCGGGTAA |
| aroGtb-A | SEQ ID NO.67 | CAGCGGCAGGTGAGTTTCTC |
| aroGtb-S | SEQ ID NO.68 | TGAGTTTCTC AATATGATCACCCCAC |
| pGRB-yjiV-s | SEQ ID NO.69 | AGTCCTAGGTATAATACTAGTGGCTGGATCGATTTCGTTCCGTTTTAGAGCTAGAA |
| pGRB-yjiV-a | SEQ ID NO.70 | TTCTAGCTCTAAAACGGAACGAAATCGATCCAGCCACTAGTATTATACCTAGGACT |
| ilvG-US | SEQ ID NO.71 | ACCGAGGAGCAGACAATGAATAA |
| ilvG-UA | SEQ ID NO.72 | AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTGATGGCAACAAC |
| ilvG-DS | SEQ ID NO.73 | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCTATCTACGCGCCGTTGTTGT |
| ilvG-DA | SEQ ID NO.74 | GCGCTGGCTAACATGAGGAA |
| talB-s | SEQ ID NO.75 | TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGACGGACAAATTGACCTCCC |
| talB-a | SEQ ID NO.76 | GATCGGCGATCTGCTGTAACAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTG |
| pGRB-ilvG-s | SEQ ID NO.77 | AGTCCTAGGTATAATACTAGTGGAAGAGTTGCCGCGCATCAGTTTTAGAGCTAGAA |
| pGRB-ilvG-a | SEQ ID NO.78 | TTCTAGCTCTAAAACTGATGCGCGGCAACTCTTCCACTAGTATTATACCTAGGACT |
| yeeL-US | SEQ ID NO.79 | TTCATCGGGACGAGTGGAGA |
| yeeL-UA | SEQ ID NO.80 | GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGCCATAGCATCGCCAATCTGATC |
| yeeL-DS | SEQ ID NO.81 | CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCCAAAGGTGAAGATAAAGCCAGG |
| yeeL-DA | SEQ ID NO.82 | CATTCCCTCTACAGAACTAGCCCT |
| pGRB-yeeL-s | SEQ ID NO.83 | AGTCCTAGGTATAATACTAGTAACACAGCAATACGGTACGCGTTTTAGAGCTAGAA |
| pGRB-yeeL-a | SEQ ID NO.84 | TTCTAGCTCTAAAACGCGTACCGTATTGCTGTGTTACTAGTATTATACCTAGGACT |
| yghE-US | SEQ ID NO.85 | GTCAGGCACTGGCGAAAGAT |
| yghE-UA | SEQ ID NO.86 | AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACGCAAGCCATAAACCCACA |
| yghE-DS | SEQ ID NO.87 | CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTCCGACATCGAAATGCGT |
| yghE-DA | SEQ ID NO.88 | GGCGTTGTTGTGGCAGATT |
| bbxfpk-S | SEQ ID NO.89 | TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGACCAACCCGGTGATTGGCA |
| bbxfpk-A | SEQ ID NO.90 | CGCGGGTGATAATGAATAACAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTG |
| pGRB-yghE-s | SEQ ID NO.91 | AGTCCTAGGTATAATACTAGTGCTGAAAAAATATCGCCCACGTTTTAGAGCTAGAA |
| pGRB-yghE-a | SEQ ID NO.92 | TTCTAGCTCTAAAACGTGGGCGATATTTTTTCAGCACTAGTATTATACCTAGGACT |
| yjgX-US | SEQ ID NO.93 | GGAAGTCAACGGGTTATGCG |
| yjgX-UA | SEQ ID NO.94 | AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAAAAATCACCACGAATACCAGAATC |
| yjgX-DS | SEQ ID NO.95 | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACAGTGTCTTCCCTGAGCCG |
| yjgX-DA | SEQ ID NO.96 | GGCGAAGGATACCATCAAGC |
| aroB-1 | SEQ ID NO.97 | TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGAGAGGATTGTCGTTACTC |
| aroB-2 | SEQ ID NO.98 | CCGATTGTCAATCAGCGTAAAGGAA |
| aroD-1 | SEQ ID NO.99 | AGGAAATGAAAACCGTAACTGTAAAAGAT |
| aroD-2 | SEQ ID NO.100 | ACTATTTTACACCAGGCATAAAGGAA |
| aroE-1 | SEQ ID NO.101 | AGGAAATGGAAACCTATGCTGTTTTTGG |
| aroE-2 | SEQ ID NO.102 | TGATATTCCGGCGATCCCGCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTG |
| pGRB-yjgX-s | SEQ ID NO.103 | AGTCCTAGGTATAATACTAGTTCGCGACCACCGTAACTGGCGTTTTAGAGCTAGAA |
| pGRB-yjgX-a | SEQ ID NO.104 | TTCTAGCTCTAAAACGCCAGTTACGGTGGTCGCGAACTAGTATTATACCTAGGACT |
| ylbE-US | SEQ ID NO.105 | ACCCAACCTTACGCAACCAG |
| ylbE-UA | SEQ ID NO.106 | AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATTGTTCGATAACCGCAGCAT |
| ylbE-DS | SEQ ID NO.107 | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCGCTGGCGTGCTTTGAA |
| ylbE-DA | SEQ ID NO.108 | GGCGTAACTCAGCAGGCAG |
| aroK-1 | SEQ ID NO.109 | TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGCAGAGAAACGCAATATCT |
| aroK-2 | SEQ ID NO.110 | ACATGCTGGAAAGCAACTAAAGGAA |
| aroA-1 | SEQ ID NO.111 | AGGAAATGGAATCCCTGACGTTACA |
| aroA-2 | SEQ ID NO.112 | GCCAGGCAGCCTGAAGGAA |
| aroC-1 | SEQ ID NO.113 | AGGAAATGGCTGGAAACACAATTG |
| aroC-2 | SEQ ID NO.114 | TGATATTCCACGCTGGTAACAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTG |
| pGRB-ylbE-s | SEQ ID NO.115 | AGTCCTAGGTATAATACTAGTACACTGGCTGGATGTGCAACGTTTTAGAGCTAGAA |
| pGRB-ylbE-a | SEQ ID NO.116 | TTCTAGCTCTAAAACGTTGCACATCCAGCCAGTGTACTAGTATTATACCTAGGACT |
| yjiV-US | SEQ ID NO.117 | TGTGACTGTGGAAGCCCTGTAT |
| yjiV-UA | SEQ ID NO.118 | TTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAATTCGGGCTGTCCCTTGTC |
| yjiV-DS | SEQ ID NO.119 | GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGTGGCACCTGAATGACGAACT |
| yjiV-DA | SEQ ID NO.120 | TGGCGACATTCCCTTCCTT |
| TrpEfbr-1 | SEQ ID NO.121 | TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCAAACACAAAAACCGACTCT |
| TrpD-2 | SEQ ID NO.122 | CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACCCTCGTGCCGCCAG |
| pGRB-yjiV-s | SEQ ID NO.123 | AGTCCTAGGTATAATACTAGTACACTGGCTGGATGTGCAACGTTTTAGAGCTAGAA |
| pGRB-yjiV-a | SEQ ID NO.124 | TTCTAGCTCTAAAACGGAACGAAATCGATCCAGCCACTAGTATTATACCTAGGACT |
| yjiT-US | SEQ ID NO.125 | AATAGTTGTTGCCGCCTGAGT |
| yjiT-UA | SEQ ID NO.126 | TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAAAAACAGGCAGCAAAGTCCC |
| yjiT-DS | SEQ ID NO.127 | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATAAGCACTACCTGTGAAGGGATGT |
| yjiT-DA | SEQ ID NO.128 | CAGGGCTTCCACAGTCACAAT |
| TrpD-1 | SEQ ID NO.129 | TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGCTGACATTCTGCTGCTC |
| TrpC-2 | SEQ ID NO.130 | CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAATATGCGCGCAGCGTC |
| pGRB-yjiT-s | SEQ ID NO.131 | AGTCCTAGGTATAATACTAGTAGGGATTATGAACGGCAATGGTTTTAGAGCTAGAA |
| pGRB-yjiT-a | SEQ ID NO.132 | TTCTAGCTCTAAAACCATTGCCGTTCATAATCCCTACTAGTATTATACCTAGGACT |
| yjiP-US | SEQ ID NO.133 | GCCATACCGCCAGCAAGAT |
| yjiP-UA | SEQ ID NO.134 | AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGCAGATATTCCCCTTTCCACC |
| yjiP-DS | SEQ ID NO.135 | aaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatGACGGATGACAAACGCAAAGC |
| yjiP-DA | SEQ ID NO.136 | AAAGGCGGATTTTTACTGTGGA |
| TrpB-1 | SEQ ID NO.137 | TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGACAACATTACTTAACCCCTATTTTG |
| TrpA-2 | SEQ ID NO.138 | CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAACTGCGCGTCGCCGC |
| pGRB-yjiP-s | SEQ ID NO.139 | AGTCCTAGGTATAATACTAGTCTTTGTCGATGAAAAATTGCGTTTTAGAGCTAGAA |
| pGRB-yjiP-a | SEQ ID NO.140 | TTCTAGCTCTAAAACGCAATTTTTCATCGACAAAGACTAGTATTATACCTAGGACT |
| ycdN-US | SEQ ID NO.141 | GATTTTGACGCCACCAACACC |
| ycdN-UA | SEQ ID NO.142 | GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCAATCCACATCACACAATCCATC |
| ycdN-DS | SEQ ID NO.143 | CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAAGGGATTTTTGGCTATCAGG |
| ycdN-DA | SEQ ID NO.144 | CATATCGTATTCGCCAGGCTG |
| serA-1 | SEQ ID NO.145 | ATGGCAAAGGTATCGCTGGAGA |
| serA-344-2 | SEQ ID NO.146 | GGCGTCGTCTGATGCACATC |
| serA-346-3 | SEQ ID NO.147 | GATGCACATCGCGGAAGCGCGTCCGGGC |
| serA-4 | SEQ ID NO.148 | GCGCCCGTCTGCTGTACTAA |
| pGRB-ycdN-s | SEQ ID NO.149 | AGTCCTAGGTATAATACTAGTCATTACTGCGTGAAGGCGCGGTTTTAGAGCTAGAA |
| pGRB-ycdN-a | SEQ ID NO.150 | TTCTAGCTCTAAAACCGCGCCTTCACGCAGTAATGACTAGTATTATACCTAGGACT |
| mbhA-US | SEQ ID NO.151 | GCCAGCACGAACATAATCCC |
| mbhA-UA | SEQ ID NO.152 | GGTCTGTTTCCTGCTAGCACTATACCTAGGACTGAGCTAGCCGTAAACACGGTGGCAGGTTTTGG |
| mbhA-DS | SEQ ID NO.153 | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCAAAAGTGCGTCCGATAC |
| mbhA-DA | SEQ ID NO.154 | CGGCGTAATCACAAACTGGC |
| glnA-3 | SEQ ID NO.155 | TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGCAAAGTACACTAGAGAAGATATCG |
| glnAtb-A | SEQ ID NO.156 | CGACAAAGGCGGATATTTCGAC |
| glnAtb-S | SEQ ID NO.157 | TTTCGACATCGCTCCAACTGATTTAGG |
| glnA-4 | SEQ ID NO.158 | CACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAATACTGAGACATATACTGTTCGCGT |
| pGRB-mbhA-s | SEQ ID NO.159 | TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACACGGTGGCAGGTTTTGG |
| pGRB-mbhA-a | SEQ ID NO.160 | AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACCAAAAGTGCGTCCGATAC |
| rph-US | SEQ ID NO.161 | ATAGCGCAGGGTACATTCCACT |
| rph-UA | SEQ ID NO.162 | AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCTTCTTCAATAGAGGCGGTACA |
| rph-DS | SEQ ID NO.163 | aaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatTGCCGCAGAGACCGACAT |
| rph-DA | SEQ ID NO.164 | ACAGCGGTTGTGGTGGCA |
| yddG-1 | SEQ ID NO.165 | TCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGACACGACAAAAAGCAACGC |
| yddG-2 | SEQ ID NO.166 | GGCGACACGTCGTGGTTAACAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTG |
| pGRB-rph-s | SEQ ID NO.167 | AGTCCTAGGTATAATACTAGTGGCTGGATCACCGCAGAGTAGTTTTAGAGCTAGAA |
| pGRB-rph-a | SEQ ID NO.168 | TTCTAGCTCTAAAACTACTCTGCGGTGATCCAGCCACTAGTATTATACCTAGGACT |
In the examples, L-tryptophan-producing strains were constructed by the following methods:
(1) Constructing chassis strains: taking E.coli W3110 as an original strain, knocking out the L-tryptophan degradation pathway gene: tnaA; knocking out repressor regulatory protein genes tyrR, trpR; introducing a 3-deoxy-7-phosphoheptanoic acid synthase gene after the feedback, and replacing trpLE genes in a tryptophan operon with P trc-trpEfbr genes;
(2) Strengthening the supply of the aromatic precursor erythrose 4-phosphate: integrating the transaldolase gene talB in a double copy form in the genome; the phosphoketolase gene bbxfk derived from bifidobacterium breve is introduced in the heterologous way, and the genes are all transcribed by using trc promoter;
(3) Strengthening shikimic acid and branching acid pathway carbon flux: the shikimic acid and key enzymes of the branch acid pathway are expressed in series to construct the artificial operons aroB-aroD-aroE and aroK-aroA-aroC, and the transcription of the artificial operons is enhanced through the trc promoter;
(4) Optimizing tryptophan operon expression: the tryptophan operon TrpE fbr DCBA was split into three artificial operons TrpE fbr D, trpDC, trpBA and integrated into pseudogene sites yjiV, yjiT, yjiP, all using trc promoters to enhance transcription;
(5) Improving serine and glutamine supply in tryptophan synthesis pathway: serine is one of direct precursors for synthesizing tryptophan, and glutamine provides amino for synthesizing tryptophan, so that a glutamine synthesis enzymolysis feedback gene glnA fbr from bacillus subtilis is introduced into a genome over-expressed D-3-phosphoglycerate dehydrogenation enzymolysis feedback gene serA fbr in a heterologous manner, and the genes are all transcribed by using a trc promoter;
(6) Enhancing L-tryptophan output ability: the gene yddG of the aromatic export protein is overexpressed in the genome in double copy form, and both genes are transcribed using the trc promoter.
The term "the present invention" refers to in reference to 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.）,, the method for editing genes used in the above-mentioned genetic manipulation, and unless otherwise noted, the term is explained in the literature. The "knockout" refers to the inactivation of a target gene, and the "introduction" refers to the insertion of an exogenous gene into the genome of an engineering bacterium after the linkage of the exogenous gene with a promoter and a terminator.
Example 1
This example is intended to illustrate the specific construction procedure of strain YTP 17. In particular, in the embodiment, if there is a method for operating the same type of gene, only 1 time is provided and annotation is made, and no redundant description is given.
<1> Knockout of tnaA Gene: taking E.coli W3110 genome as a template, respectively taking tnaA-US, tnaA-UA, tnaA-DS and tnaA-DA, amplifying by pcr to obtain an upstream homology arm and a downstream homology arm, and then taking the upstream homology arm and the downstream homology arm as templates, taking tnaA-US and tnaA-DA as primers, and amplifying by overlapping pcr to obtain an overlapped fragment; taking pGRB-tnaA-s and pGRB-tnaA-a as primers, annealing to obtain a gRNA fragment, and connecting the gRNA fragment with a pGRB vector to obtain tnaA-pGRB; e.coli W3110 electrotransformation competent cells were prepared, overlapping fragments were electrotransformed into competent cells together with tnaA-pGRB, and positive transformants were selected to obtain strain YTP01.
<2> Knock-out tyrR gene: the same procedure as in <1> was followed except that the primers used were tyrR-US, tyrR-UA, tyrR-DS, tyrR-DA, pGRB-tyrR-s, pGRB-tyrR-a. Competent cells were YTP01 to obtain strain YTP02.
<3> Knockout trpR gene: the same procedure as in <1> was followed except that the primers used were trpR-US, trpR-UA, trpR-DS, trpR-DA, pGRB-trpR-s, pGRB-trpR-a. Competent cells were YTP02, strain YTP03 was obtained.
<4> Knock-out trpLE gene and integration of Ptrc-trpE fbr at this site: the E.coli W3110 genome is taken as a template, DNA sequences of exogenous cleavage sites are added on primers trpLE-2 and trpLE-3 in a segmented manner, primers trpLE-1 and trpLE-2, trpLE-3 and trpLE-4 are respectively amplified by PCR to obtain an upstream homology arm and a downstream homology arm, the recovered upstream homology arm and the recovered downstream homology arm are taken as templates, and overlapping PCR is carried out by using primers trpLE-1 and trpLE-4 to obtain a copy piece segment which is required for knocking out the gene trpLE and has the exogenous cleavage sites, pGRB-LE-s and pGRB-LE-a are taken as primers, annealing is carried out to obtain a gRNA segment, and the gRNA segment is connected with a pGRB vector to obtain pGRB-LE; preparing YTP03 electrotransformation competent cells, performing electrotransformation on target fragments and pGRB-LE together into competent cells, and screening to obtain positive transformants to obtain a strain YTP04;
Then, using Escherichia coli W3110 as a template, carrying out PCR amplification by using primers trpE-1 and trpE-2, trpE-5 and trpE-6 to obtain upstream and downstream homology arms, designing a mutant nucleotide sequence on trpEtb-A, trpEtb-S and having homology sequences, carrying out PCR amplification by using primers trpE-3/trpEtb-A and trpEtb-S/trpE-4 to obtain a fragment 1 and a fragment 2 respectively, and carrying out overlap PCR by using the primers trpE-3/trpE-4 to obtain a target fragment P trc-trpEfbr by using the fragments 1 and 2 as templates; taking pGRB-1-s and pGRB-1-a as primers, annealing to obtain gRNA fragments, and connecting the gRNA fragments with a pGRB vector to obtain pGRB-1; preparing YTP04 electrotransformation competent cells, electrotransforming target fragments into competent cells together with pGRB-1, and screening to obtain positive transformants to obtain the strain YTP05.
<5> Control of aroG fbr gene overexpression using trc promoter at yjiV pseudogene locus: the E.coli W3110 genome is used as a template, yjiV-US/yjiV-UA and yjiV-DS/yjiV-DA are respectively used for amplification to obtain an upstream homology arm and a downstream homology arm, then, mutant nucleotide sequences are designed on aroGtb-A and aroGtb-S and have homology sequences, primers aroG-3/aroGtb-A and aroGtb-S/aroG-4 are respectively used for PCR amplification to obtain a fragment 1 and a fragment 2, and then the fragment 1 and the fragment 2 are used as templates, and the primers aroG-3/aroG-4 are used for overlap PCR to obtain a target fragment P trc-aroGfbr; the upstream and downstream homology arms and target gene fragments are used as templates, yjiV-US and yjiV-DA are used as primers, and overlapping pcr amplification is carried out to obtain overlapping fragments; taking pGRB-yjiV-s and pGRB-yjiV-a as primers, annealing to obtain a gRNA fragment, and connecting the gRNA fragment with a pGRB vector to obtain pGRB-yjiV; preparing YTP05 electrotransformation competent cells, electrotransforming target fragments into competent cells together with pGRB-yjiV, and screening to obtain positive transformants to obtain the strain YTP06.
<6> Control of talB Gene overexpression at ilvG pseudogene locus using trc promoter: E.coliW3110 genome is used as a template, ilvG-US/ilvG-UA and ilvG-DS/ilvG-DA and talk-s/talk-a are respectively amplified by pcr to obtain an upstream homology arm, a downstream homology arm and a target gene fragment, and then the upstream homology arm, the downstream homology arm and the target gene fragment are used as templates, ilvG-US and ilvG-DA are used as primers, and overlapped pcr is used for amplification to obtain overlapped fragments; taking pGRB-ilvG-s and pGRB-ilvG-a as primers, annealing to obtain a gRNA fragment, and connecting the gRNA fragment with a pGRB vector to obtain pGRB-ilvG; preparing YTP06 electrotransformation competent cells, electrotransforming target fragments and pGRB-ilvG into competent cells together, and screening to obtain positive transformants to obtain the strain YTP07.
<7> Control of talB gene overexpression using trc promoter at yeeL pseudogene locus: the same procedure as in <6> was followed except that yeeL-US, yeeL-UA, yeeL-DS, yeeL-DA, pGRB-yeeL-s, pGRB-yeeL-a were used as primers. Competent cells were YTP07 to obtain strain YTP08.
<8> Control of bbxfpk gene overexpression using the Ptrc promoter at yghE pseudogene locus: the same procedure as in <6> was followed except that the bbxfpk gene was synthesized and codon optimized by Jin Weizhi Biotechnology Inc., using the primers yghE-US, ygHE-UA, ygHE-DS, ygHE-DA, bbxfpk-S, bbxfpk-A, pGRB-yghE-s, pGRB-yghE-a. Competent cells were YTP08, strain YTP09 was obtained.
<9> Expression of human operon aroBDE was controlled using trc promoter at yjgX pseudogene locus: the E.coli W3110 genome is used as a template, primers yjgX-US/yjgX-UA and yjgX-DS/yjgX-DA are respectively used for obtaining upstream and downstream homology arms through pcr amplification, and primers aroB-1/aroB-2, aroD-1/aroD-2 and aroE-1/aroE-2 are used for performing pcr amplification to obtain aroB, aroD and aroE fragments, wherein aroB-2 and aroD-1 have homology sequences, and aroD-2 and aroE-1 have homology sequences; overlapping pcr with aroB, aroD and aroE fragments as templates and primers aroB-1 and aroE-2 to obtain trc-aroBDE fragments; the upstream and downstream homology arms and the target gene fragment are used as templates, yjgX-US and yjgX-DA are used as primers, and overlapping pcr amplification is carried out to obtain overlapping fragments; taking pGRB-yjgX-s and pGRB-yjgX-a as primers, annealing to obtain a gRNA fragment, and connecting the gRNA fragment with a pGRB vector to obtain pGRB-yjgX; preparing YTP09 electrotransformation competent cells, electrotransforming target fragments into competent cells together with pGRB-yjgX, and screening to obtain positive transformants to obtain the strain YTP10.
<10> Expression of human operon aroKAC was controlled using the Ptrc promoter at ylbE pseudogene site: the same procedure as in <9> was followed except that ylbE-US/ylbE-UA, ylbE-DS/ylbE-DA, aroK-1/aroK-2, aroA-1/aroA-2, aroC-1/aroC-2, pGRB-ylbE-s, pGRB-ylbE-a were used as primers. Competent cells were YTP10 to obtain strain YTP11.
<11> Expression of the human operon TrpE fbr D was controlled at yjiV pseudogene locus using bba_j23101 promoter: the E.coli W3110 genome is used as a template, the primers yjiV-US/yjiV-UA and yjiV-DS/yjiV-DA are respectively used for obtaining an upstream homology arm and a downstream homology arm through pcr amplification, the strain YTP05 genome is used as a template, and the primers TrpE fbr -1 and TrpD-2 are used for obtaining a trc-TrpE fbr D fragment through pcr amplification. Taking pGRB-yjiV-s and pGRB-yjiV-a as primers, annealing to obtain a gRNA fragment, and connecting the gRNA fragment with a pGRB vector to obtain pGRB-yjiV; preparing YTP11 electrotransformation competent cells, electrotransforming target fragments into competent cells together with pGRB-yjiV, and screening to obtain positive transformants to obtain the strain YTP12.
<12> Expression of human operon TrpDC was controlled at yjiT pseudogene locus using bba_j23101 promoter: the same procedure as in <11> was followed except that yjiT-US/yjiT-UA, yjiT-DS/yjiT-DA, trpD-1, trpC-2, pGRB-yjiT-s, pGRB-yjiT-a were used as primers. Competent cells were YTP12 to obtain strain YTP13.
<13> Expression of human operon TrpBA was controlled at yjiP pseudogene locus using bba_j23101 promoter: the same procedure as in <11> was followed except that yjiP-US/yjiP-UA, yjiP-DS/yjiP-DA, trpB-1, trpA-2, pGRB-yjiP-s, pGRB-yjiP-a were used as primers. The competent cell was YTP13 to obtain strain YTP14.
<14> Control of serA fbr Gene overexpression at ycdN pseudogene locus using trc promoter: the same procedure as in <5> was followed except that ycdN-US/ycdN-UA, ycdN-DS/ycdN-DA, serA-1, serA-344-2, serA-346-3, serA-4, pGRB-ycdN-s, pGRB-ycdN-a were used as primers. The competent cell was YTP14 to obtain strain YTP15.
<15> GlnA fbr gene overexpression was controlled using trc promoter at mbhA pseudogene locus: the same procedure as in <5> was followed except that mbhA-US/mbhA-UA, mbhA-DS/mbhA-DA, glnA-3/glnAtb-A, glnAtb-S/glnA-4, pGRB-mbhA-S, pGRB-mbhA-a were used. Competent cells were YTP15 to obtain strain YTP16.
<16> Control of yddG Gene overexpression at the rph pseudogene locus using trc promoter: the same procedure as in <6> was followed except that the primers used were rph-US, rph-UA, rph-DS, rph-DA, yddG-1, yddG-2, pGRB-yddG-s, pGRB-yddG-a. Competent cells were YTP16 to obtain strain YTP17.
Example 2
The strain YTP17 described in example 1 was used for fermentation production of L-tryptophan in a 5L fermenter, and the specific steps are as follows:
(1) Slant culture: inoculating L-tryptophan producing strain on slant culture medium as first generation slant culture, and culturing at 36-37deg.C for 13 hr; the slant culture medium is a universal LB solid culture medium; inoculating the colony of the first generation slant culture to a new slant culture medium as a second generation slant culture, and culturing at 36-37 ℃ for 12h;
(2) Seed culture: washing the second generation inclined plane with sterile water to a seed culture medium, wherein the culture temperature is 36 ℃, and controlling the automatic feeding of 25% ammonia water solution through a 5L bioreactor to maintain the pH value of seed liquid at 7.0+/-0.2 and the dissolved oxygen value at 30-50%, and performing the next fermentation culture when the OD 600nm of the seed liquid reaches 15, wherein the adopted seed culture medium is as follows: 30 g/L of glucose, 5g/L of yeast extract powder, 3 g/L of ammonium sulfate, 4 g/L of monopotassium phosphate, 1 g/L of anhydrous magnesium sulfate, 20mg/L of ferrous sulfate heptahydrate, 20mg/L of manganese sulfate monohydrate, 0.5 mg/L of V H1 mg/L,VB1 and the balance of water;
(3) Fermentation culture: the inoculation amount is 20%, the culture temperature is 37 ℃, the pH of seed liquid is maintained at 7.0+/-0.2 by controlling automatic feeding of 25% ammonia water solution through a bioreactor, and the dissolved oxygen value is 30% -50%, wherein the adopted fermentation culture medium is as follows: glucose 20 g/L, yeast extract 5 g/L, ammonium sulfate 3 g/L, monopotassium phosphate 4 g/L, anhydrous magnesium sulfate 1.5 g/L, ferrous sulfate heptahydrate 20 mg/L, manganese sulfate monohydrate 20 mg/L, V H0.5 mg/L,VB1 1 mg/L, and the balance water; after the glucose in the culture medium is consumed, 80% (m/v) glucose solution is fed in, and the glucose concentration in the fermentation culture medium is maintained at 0.1-1g/L, and the fermentation period is 40 h.
As shown in FIG. 1, the yield of L-tryptophan reaches 52.3g/L after fermentation in the 5L fermenter for 40 h, and the sugar acid conversion rate is 20%. In this example, a 5L fermenter is used, which is small, and as known to those skilled in the art, the sugar acid conversion rate will stabilize at 21% -22% in the case of large scale production, which has more obvious technical advantages.
The foregoing is merely illustrative of the preferred embodiments of this invention, and it will be appreciated by those skilled in the art that variations and modifications of the invention and strain changes, which are carried out by or based on the methods of this invention, may be made without departing from the spirit of this invention.
Claims (8)
1. A genetically engineered bacterium for producing L-tryptophan is characterized in that: e.coli W3110 is taken as an original strain, tnaA, tyrR, trpR genes are deleted, the transcription level of trpE fbr、aroGfbr、serAfbr and talB, aroB, aroD, aroE, aroK, aroA, aroC, yddG genes is up-regulated, and each gene cluster TrpE fbr D in TrpE fbr DCBA of tryptophan operon is reinforced, Expression of TrpDC and TrpBA, heterologous introduction of phosphoketolase gene bbxfpk from bifidobacterium breve and glnA fbr from bacillus subtilis; Wherein the starting strain E.coli W3110 is E.coli W3110 ATCC 27325, and the nucleotide sequence of the tnaA gene is shown in a sequence table SEQ ID NO. 5; the nucleotide sequence of the tyrR gene is shown in a sequence table SEQ ID NO. 6; the nucleotide sequence of the trpR gene is shown in a sequence table SEQ ID NO. 7; the nucleotide sequence of trpE fbr is shown in a sequence table SEQ ID NO. 8; The nucleotide sequence of aroG fbr is shown in a sequence table SEQ ID NO. 9; the nucleotide sequence of the talB gene is shown in a sequence table SEQ ID NO. 10; the nucleotide sequence of bbxfpk gene is shown in sequence table SEQ ID NO. 11; the nucleotide sequence of aroB gene is shown in sequence table SEQ ID NO. 12; the nucleotide sequence of aroD gene is shown in sequence table SEQ ID NO. 13; the nucleotide sequence of aroE gene is shown in sequence table SEQ ID NO. 14; The nucleotide sequence of aroK gene is shown in sequence table SEQ ID NO. 16; the nucleotide sequence of aroA gene is shown in sequence table SEQ ID NO. 17; the nucleotide sequence of aroC gene is shown in sequence table SEQ ID NO. 18; the nucleotide sequence of the tryptophan operon TrpE fbr DCBA is shown in a sequence table SEQ ID NO. 20; the nucleotide sequence of the mini-operon TrpE fbr D is shown in a sequence table SEQ ID NO. 21; The nucleotide sequence of the mini-operon TrpDC is shown in a sequence table SEQ ID NO. 22; the nucleotide sequence of the mini-operon TrpBA is shown in a sequence table SEQ ID NO. 23; the nucleotide sequence of the serA fbr gene is shown in a sequence table SEQ ID NO. 24; the nucleotide sequence of the glnA fbr gene is shown in a sequence table SEQ ID NO. 25; the nucleotide sequence of the yddG gene is shown in a sequence table SEQ ID NO. 26.
2. The genetically engineered bacterium for producing L-tryptophan according to claim 1, wherein: taking E.coli W3110 as an original strain, knocking out tnaA, tyrR, trpR genes; the trpE fbr gene transcription is enhanced by using a trc promoter, and is integrated into a trpLE gene locus; the trc promoter is utilized to strengthen aroG fbr gene transcription and is integrated into yjiV pseudogene locus; the trc promoter is utilized to strengthen the transcription of the talB gene and is respectively integrated into ilvG and yeeL pseudogene loci in a double copy form in the genome; the trc promoter is utilized to strengthen the transcription of the phosphoketolase gene bbxfpk derived from bifidobacterium breve and is integrated into the yghE pseudogene locus; the trc promoter is used for strengthening aroB, aroD and aroE gene transcription and is integrated into yjgX pseudogene sites to form a mini-operon; the trc promoter is used for strengthening aroK, aroA and aroC gene transcription and is integrated into ylbE pseudogene sites to form a mini-operon; splitting the modified tryptophan operon TrpE fbr DCBA into three micromanipulators TrpE fbr D, trpDC and TrpBA, and integrating the three micromanipulators into yjiV, yjiT and yjiP pseudogene loci by utilizing a BBa_j23101 promoter; the trc promoter is utilized to strengthen the transcription of the serA fbr gene and is integrated into a ycdN pseudogene locus; the trc promoter is utilized to strengthen the transcription of the glnA fbr gene from bacillus subtilis and is integrated into mbhA pseudogene locus; the trc promoter is used to enhance the transcription of the yddG gene and is integrated in the genome in double copies at the rph and yjiK pseudogene sites, respectively.
3. The genetically engineered bacterium for producing L-tryptophan according to claim 1 or 2, wherein: the nucleotide sequence of the trc promoter is shown in a sequence table SEQ ID NO. 1; the nucleotide sequence of the BBa_j23101 promoter is shown as a sequence table SEQ ID NO. 2.
4. The method for constructing a genetically engineered bacterium for producing L-tryptophan according to any one of claims 1 to 3, wherein: the method comprises the following specific steps:
(1) Constructing chassis strains: taking E.coli W3110 as an original strain, and knocking out an L-tryptophan degradation pathway gene tnaA; knocking out repressor regulatory protein gene tyrR and trpR; introducing a 3-deoxy-7-phosphoheptanoic acid synthase gene aroG fbr after the solution feedback, and inserting a trpLE gene in a tryptophan operon as a pseudogene locus into a trpE fbr gene;
(2) Strengthening the supply of the aromatic precursor erythrose 4-phosphate: integrating the transaldolase gene talB in a double copy form in the genome; the phosphoketolase gene bbxfk derived from bifidobacterium breve is introduced in the heterologous way, and the genes are all transcribed by using trc promoter;
(3) Strengthening shikimic acid and branching acid pathway carbon flux: carrying out tandem expression on shikimic acid and key enzymes of a branched acid pathway, constructing artificial operons aroBDE and aroKAC, and strengthening transcription of the artificial operons through a trc promoter;
(4) Optimizing tryptophan operon expression: the tryptophan operon TrpE fbr DCBA was split into three artificial operons TrpE fbr D, trpDC, trpBA and integrated into pseudogene sites yjiV, yjiT, yjiP, respectively, all using trc promoter to enhance transcription;
(5) Improving serine and glutamine supply in tryptophan synthesis pathway: the method comprises the steps of introducing a bacillus subtilis-derived glutamine synthetic enzymolysis feedback gene glnA fbr into a genome overexpression D-3-phosphoglycerate dehydrogenation enzymolysis feedback gene serA fbr in a heterologous manner, and using trc promoters to strengthen transcription of the genes;
(6) Enhancing L-tryptophan output ability: the gene yddG of the aromatic export protein is overexpressed in the genome in double copy form, and both genes are transcribed using the trc promoter.
5. Use of the genetically engineered bacterium for producing L-tryptophan according to one of claims 1 to 3 for the fermentative production of L-tryptophan.
6. The use of the genetically engineered bacterium for producing L-tryptophan according to claim 5, wherein: the fermentation is carried out by using a mechanical stirring type fermentation tank, and the specific steps are as follows:
(1) Slant culture: inoculating L-tryptophan producing strain on slant culture medium as first generation slant culture, and culturing at 36-37deg.C for 12-16 hr; inoculating the colony of the first generation slant culture to a new slant culture medium as a second generation slant culture, and culturing at 36-37 ℃ for 10-12h;
(2) Seed culture: washing the second generation inclined plane with sterile water to a seed culture medium, wherein the culture temperature is 36 ℃, controlling the automatic fed-batch ammonia water solution to maintain the pH value of the seed solution at 7.0+/-0.2 through a bioreactor, and carrying out the next fermentation culture when the dissolved oxygen value of the seed solution OD 600nm reaches 15;
(3) Fermentation culture: the inoculation amount is 20%, the culture temperature is 37 ℃, the pH value of the seed solution is maintained at 7.0+/-0.2 by controlling automatic feeding of ammonia water solution through a bioreactor, and the dissolved oxygen value is 30% -50%.
7. The use of the genetically engineered bacterium for producing L-tryptophan according to claim 6, wherein: the seed culture medium in the step (2) is as follows: 20-40 g/L of glucose, 2-5 g/L of yeast extract powder, 1-5 g/L of ammonium sulfate, 1-5 g/L of monopotassium phosphate, 0.5-2 g/L of anhydrous magnesium sulfate, 10-30 mg/L of ferrous sulfate heptahydrate, 10-30 mg/L of manganese sulfate monohydrate, 0.5-1 mg/L of V H0.1-0.5 mg/L,VB1 and the balance of water.
8. The use of the genetically engineered bacterium for producing L-tryptophan according to claim 6, wherein: the fermentation medium in the step (3) is as follows: 20-40 g/L of glucose, 2-5 g/L of yeast extract powder, 1-5 g/L of ammonium sulfate, 1-5 g/L of monopotassium phosphate, 0.5-2 g/L of anhydrous magnesium sulfate, 30-60 mg/L of ferrous sulfate heptahydrate, 20-40 mg/L of manganese sulfate monohydrate and 0.5-1 mg/L of V H0.1-0.5 mg/L,VB1 .
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| CN113549588A (en) * | 2021-06-25 | 2021-10-26 | 天津科技大学 | Genetically engineered bacterium for producing 5-hydroxytryptophan and construction method and application thereof |
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| CN113549588A (en) * | 2021-06-25 | 2021-10-26 | 天津科技大学 | Genetically engineered bacterium for producing 5-hydroxytryptophan and construction method and application thereof |
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