CN116376852B - Double-peptide tripeptide permease mutant and application thereof in production of L-tryptophan - Google Patents

Double-peptide tripeptide permease mutant and application thereof in production of L-tryptophan Download PDF

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CN116376852B
CN116376852B CN202310327138.1A CN202310327138A CN116376852B CN 116376852 B CN116376852 B CN 116376852B CN 202310327138 A CN202310327138 A CN 202310327138A CN 116376852 B CN116376852 B CN 116376852B
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tryptophan
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dtpa
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卢煜
曹国强
施天元
周国旺
李靖瑶
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Harbin Xiangbai Biotechnology Co ltd
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Abstract

A dipeptide tripeptide permease mutant and application thereof in producing L-tryptophan belong to the technical field of genetic engineering. Aiming at the problem that the tryptophan is transported in cells due to the existence of permease when the gram-negative bacillus is utilized to produce the tryptophan, so that the tryptophan yield is affected, the invention obtains the high-yield tryptophan strain through mutagenesis, obtains the dipeptide tripeptide permease mutant for improving the L-tryptophan yield, and improves the tryptophan yield through expressing the mutant in escherichia coli.

Description

Double-peptide tripeptide permease mutant and application thereof in production of L-tryptophan
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a dipeptide tripeptide permease mutant and application thereof in the production of L-tryptophan.
Background
When tryptophan is produced by gram-negative bacilli, tryptophan accumulates in large amounts in the cell, and if tryptophan is not excreted from the cell in time, and the transport protein will still transport tryptophan inward, tryptophan production will be severely affected. There are many known permeases, but there are only three reported tryptophan permeases in E.coli: mtr, tnaB, aroP. The knockout or mutation can increase the permeability of cell membranes, reduce the intracellular trafficking of tryptophan and obviously improve the tryptophan yield. As the existing industrial strain of tryptophan is subjected to genetic transformation, the problem that a large amount of synthesized tryptophan is accumulated in a cell is solved, and the yield of tryptophan is affected. The tryptophan production is still relatively insufficient compared with other amino acids with higher yields, and it is still necessary to improve the tryptophan production by modifying the strain.
Disclosure of Invention
Aiming at the problem that the intracellular transportation of tryptophan is caused by the existence of permease and the tryptophan yield is further influenced when the tryptophan is produced by utilizing gram-negative bacillus, the invention provides a dipeptide tripeptide permease mutant for improving the L-tryptophan yield, wherein the dipeptide tripeptide permease mutant is any one of the following mutants:
the amino acid sequence of the mutant dtpA is shown as SEQ ID NO. 1;
the amino acid sequence of the mutant dtpB is shown in SEQ ID NO. 2.
The invention also provides a coding sequence of the dipeptide tripeptide permease mutant. .
Further defined, the coding sequence of the mutant dtpA is shown in SEQ ID NO. 3;
further defined, the coding sequence of the mutant dtpB is shown in SEQ ID NO. 4.
The invention also provides a recombinant vector containing the coding sequence.
The invention also provides recombinant bacteria containing the recombinant vector.
The invention also provides application of the dipeptide tripeptide permease mutant in producing L-tryptophan.
The invention also provides application of the coding sequence in the production of L-tryptophan.
The invention also provides application of the recombinant vector in production of L-tryptophan.
The invention also provides application of the recombinant bacterium in producing L-tryptophan.
The invention also provides a method for improving the yield of L-tryptophan, which is to express mutant dtpA in escherichia coli, or express mutant dtpB, or express mutant dtpA and mutant dtpB at the same time; the coding sequence of the mutant dtpA is shown as SEQ ID NO. 3; the coding sequence of the mutant dtpB is shown in SEQ ID NO. 4.
Drawings
FIG. 1 is a graph showing the absorbance versus tryptophan concentration;
FIG. 2 is a schematic diagram of back mutation;
FIG. 3 is a flow chart of the acquisition of a point mutant strain;
FIG. 4 is a schematic diagram of pTH18KR-PJ23119-Ptrc-PamyL-T7 terminator plasmid.
Detailed Description
The invention aims to provide a dipeptide tripeptide permease mutant and application thereof in L-tryptophan production. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the methods and applications described herein can be modified or adapted and combined to implement and utilize the technology of this invention without departing from the spirit and scope of this invention.
The present invention will be described in further detail with reference to specific embodiments, so that those skilled in the art can better understand the present invention.
The Escherichia coli used in the examples of the present invention is Escherichia coli (Escherichia coli) CGMCC No.11073.
The culture medium for culturing the escherichia coli, which is selected in the embodiment of the invention, comprises the following components: 4g/L of monopotassium phosphate, 4g/L of dipotassium phosphate, 7g/L of disodium hydrogen phosphate, 5g/L of ammonium sulfate, 0.08g/L of ferrous sulfate, 2g/L of citric acid monohydrate, 2g/L of yeast powder, 40g/L of propane sulfonic acid, 0.0013g/L of vitamin B, 0.003g/L of biotin, 0.012g/L of manganese sulfate, 1.2g/L of magnesium sulfate heptahydrate and 30g/L of glucose monohydrate.
Example 1: obtaining of dipeptide tripeptide permease mutant for improving yield of L-tryptophan
(1) Acquisition of mutation sites
Mutagenesis is carried out on the original strain, the L-tryptophan high-yield strain is obtained through high-throughput screening, and whole genome sequencing is carried out:
adding Escherichia coli preserved on the slant culture medium into 5% glycerol, scattering, and regulating pH to 0.6-0.8; placing 10 microliters on a sterile slide, uniformly smearing, setting a mutagenesis gradient of 40s to 60s, and placing the slide in an ARTP mutagenesis instrument for mutagenesis treatment; diluting with sterile water for a proper multiple, coating, diluting and coating non-mutagenized I-S tryptophan strains, culturing for 21h, culturing the single colonies with 96 deep-hole plates in high flux, culturing for 18h in 500 microlitres, manually adding the culture medium for gun discharge, and picking the colonies by high flux equipment; centrifuging the culture solution, diluting the supernatant by 6 times, adding 9 times of p-dimethylaminobenzaldehyde sulfuric acid chromogenic solution, heating at 98 ℃ for 3 minutes, adding 4 microliters of 0.5% sodium nitrite, developing at 98 ℃ for 2 minutes, taking out and cooling for 2 minutes, taking out 200 microliters of the culture solution, and carrying out ultraviolet absorption detection at 600nm by using an enzyme-labeled instrument; drawing a standard curve: sucking tryptophan standard solution 0.05g/L, 0.1g/L, 0.15g/L, 0.2g/L, 0.25g/L, 0.3g/L, and measuring OD 600 The ultraviolet absorbance at nm was 0.1713, 0.2741, 0.4346, 0.5164, 0.6281, 0.8275, respectively, and the result of plotting a standard curve with tryptophan concentration on the abscissa and absorbance on the ordinate was shown in fig. 1. OD of the original strain after cultivation 600 The value is detected, the result is shown in Table 1, after the abnormal data is removed, the average value of the acid production of the original deep pore plate of the non-mutagenized escherichia coli is calculated to be 1.22g/L according to a standard curve, and the OD of the mutagenized strain with the highest tryptophan production is calculated 600 As 1.0741, tryptophan yield was calculated to be 2.45g/L based on a standard curve, and the strain was designated as XY-Wa-F1. The professional sequencing company was commissioned to sequence the mutant strain by using two-plus-three generation whole genome sequencing, and a plurality of mutation sites were found by comparison, including substitution of asparagine at position 144 of dtpA with histidine and substitution of valine at position 39 of homologous dtpB with glycine.
The amino acid sequence of the mutant dtpA is shown in SEQ ID NO. 1:
VSTANQKPTESVSLNAFKQPKAFYLIFSIELWERFGYYGLQGIMAVYLVKQLGMSEADSITLFSSFSALVYGLVAIGGWLGDKVLGTKRVIMLGAIVLAIGYALVAWSGHDAGIVYMGMAAIAVGNGLFKANPSSLLSTCYEKHDPRLDGAFTMYYMSVNIGSFFSMIATPWLAAKYGWSVAFALSVVGLLITIVNFAFCQRWVKQYGSKPDFEPINYRNLLLTIIGVVALIAIATWLLHNQEVARMALGVVAFGIVVIFGKEAFAMKGAARRKMIVAFILMLEAIIFFVLYSQMPTSLNFFAIRNVEHSILGLAVEPEQYQALNPFWIIIGSPILAAIYNKMGDTLPMPTKFAIGMVMCSGAFLILPLGAKFASDAGIVSVSWLVASYGLQSIGELMISGLGLAMVAQLVPQRLMGFIMGSWFLTTAGANLIGGYVAGMMAVPDNVTDPLMSLEVYGRVFLQIGVATAVIAVLMLLTAPKLHRMTQDDAADKAAKAAVA；
the amino acid sequence of the mutant dtpB is shown in SEQ ID NO. 2:
MNTTTPMGMLQQPRPFFMIFFVELWERFGYYGVQGVLAGFFVKQLGFSQEQAFVTFGAFAALVYGLISIGGYVGDHLLGTKRTIVLGALVLAIGYFMTGMSLLKPDLIFIALGTIAVGNGLFKANPASLLSKCYPPKDPRLDGAFTLFYMSINIGSLIALSLAPVIADRFGYSVTYNLCGAGLIIALLVYIACRGMVKDIGSEPDFRPMSFSKLLYVLLGSVVMIFVCAWLMHNVEVANLVLIVLSIVVTIIFFRQAFKLDKTGRNKMFVAFVLMLEAVVFYILYAQMPTSLNFFAINNVHHEILGFSINPVSFQALNPFWVVLASPILAGIYTHLGNKGKDLSMPMKFTLGMFMCSLGFLTAAAAGMWFADAQGLTSPWFIVLVYLFQSLGELFISALGLAMIAALVPQHLMGFILGMWFLTQAAAFLLGGYVATFTAVPDNITDPLETLPVYTNVFGKIGLVTLGVAVVMLLMVPWLKRMIATPESH；
the nucleotide sequence of the coding mutant dtpA is shown in SEQ ID NO. 3:
GTGTCCACTGCAAACCAAAAACCAACTGAAAGCGTCAGTTTGAACGCTTTCAAACAACCGAAGGCGTTCTATCTCATCTTCTCGATTGAGTTATGGGAACGTTTTGGTTATTACGGCCTACAAGGAATTATGGCTGTTTACCTGGTTAAACAACTGGGTATGTCTGAAGCGGATTCAATCACCCTTTTCTCTTCCTTTAGTGCCCTGGTTTATGGTCTGGTCGCTATCGGCGGCTGGTTAGGTGACAAGGTACTGGGTACTAAACGCGTAATTATGCTCGGCGCTATTGTGCTGGCGATTGGTTATGCTCTGGTTGCCTGGTCTGGTCACGACGCCGGTATCGTTTATATGGGTATGGCGGCTATTGCGGTCGGTAACGGCCTGTTTAAAGCTAACCCGTCTTCTCTGCTTTCTACATGCTATGAGAAAAACGACCCGCGTCTGGACGGTGCATTCACCATGTACTACATGTCCGTCAACATCGGCTCTTTCTTCTCTATGATTGCTACGCCGTGGCTGGCCGCGAAATACGGCTGGAGTGTTGCGTTTGCGTTGAGCGTTGTAGGCCTGCTGATCACTATCGTTAACTTCGCCTTCTGCCAACGCTGGGTTAAACAGTACGGTTCAAAACCAGACTTCGAGCCTATCAACTACCGTAACCTGCTGCTGACCATTATTGGTGTTGTGGCACTGATCGCTATCGCCACCTGGCTGCTGCACAATCAGGAAGTTGCGCGTATGGCGCTGGGCGTTGTTGCCTTCGGTATCGTGGTTATCTTCGGTAAAGAAGCCTTCGCGATGAAAGGTGCTGCGCGTCGTAAAATGATCGTTGCCTTCATCCTGATGCTCGAAGCCATTATCTTCTTCGTGCTGTACAGCCAGATGCCAACGTCACTGAACTTCTTTGCGATTCGTAACGTTGAGCACTCCATTCTGGGTCTGGCCGTAGAACCTGAGCAGTATCAGGCACTGAACCCGTTCTGGATCATCATCGGTAGTCCGATTCTGGCCGCTATCTATAACAAGATGGGCGATACCCTGCCGATGCCAACCAAGTTTGCAATCGGCATGGTGATGTGTTCTGGTGCGTTCCTGATTCTGCCGCTGGGTGCGAAATTCGCGTCTGACGCTGGTATCGTGTCTGTAAGCTGGCTGGTCGCAAGCTATGGCCTGCAGAGCATCGGGGAACTGATGATCTCTGGTCTGGGTCTGGCAATGGTTGCTCAACTCGTTCCGCAGCGTCTGATGGGCTTCATTATGGGTAGCTGGTTCCTGACCACTGCCGGTGCAAACCTGATTGGTGGTTATGTTGCGGGTATGATGGCTGTGCCGGATAACGTTACCGATCCGCTGATGTCACTGGAAGTCTATGGTCGCGTATTCTTGCAGATTGGTGTCGCTACTGCCGTTATTGCAGTACTGATGCTGCTGACCGCGCCGAAACTGCACCGCATGACGCAGGATGACGCTGCAGACAAAGCGGCGAAAGCAGCCGTAGCGTAA；
the nucleotide sequence of the coding mutant dtpB is shown in SEQ ID NO. 4:
ATGAATACAACAACACCCATGGGGATGCTGCAGCAACCTCGCCCATTTTTCATGATCTTTTTTGTCGAGTTATGGGAGCGATTCGGCTACTACGGCGTGCAGGGCGTACTGGCGGGTTTCTTCGTTAAACAGCTTGGATTCTCGCAAGAGCAGGCTTTTGTCACTTTTGGTGCTTTTGCTGCGCTGGTCTATGGCCTCATTTCCATTGGCGGCTATGTCGGCGACCACCTGCTGGGGACCAAACGCACCATTGTTCTTGGAGCACTTGTGCTGGCGATTGGCTACTTCATGACCGGCATGTCGCTACTTAAGCCTGACCTGATTTTCATCGCCCTGGGGACTATCGCTGTCGGTAACGGCCTGTTTAAAGCTAACCCAGCCAGCTTGCTTTCGAAGTGCTATCCGCCGAAAGATCCGCGGCTTGATGGCGCATTCACCCTGTTCTATATGTCGATCAACATCGGCTCGTTGATAGCGTTATCGCTGGCCCCTGTGATCGCTGATAGATTCGGTTATTCAGTCACCTACAACCTGTGCGGGGCGGGGTTAATTATCGCATTACTGGTTTACATCGCCTGTCGTGGAATGGTGAAAGACATTGGTTCTGAACCCGACTTCCGGCCAATGAGCTTCAGCAAACTGTTGTACGTGTTACTTGGCAGCGTGGTGATGATCTTCGTATGCGCATGGCTGATGCACAACGTAGAAGTCGCCAATCTGGTGCTGATTGTTCTCTCCATCGTCGTCACCATCATCTTCTTTCGTCAGGCATTCAAGCTGGATAAAACCGGGCGCAATAAAATGTTTGTCGCCTTTGTCCTGATGCTCGAAGCGGTGGTGTTTTACATTCTCTACGCCCAGATGCCAACATCGCTGAACTTCTTTGCCATCAACAACGTGCATCATGAAATTCTCGGTTTTTCCATCAACCCGGTCAGCTTCCAGGCGCTTAACCCGTTCTGGGTGGTACTCGCCAGCCCAATACTGGCAGGCATTTACACGCATCTGGGTAACAAAGGCAAAGACCTCTCGATGCCGATGAAATTTACTCTCGGCATGTTTATGTGCTCACTGGGCTTTTTGACGGCGGCAGCTGCGGGAATGTGGTTTGCGGATGCACAAGGGCTGACATCGCCATGGTTTATCGTGCTGGTGTACTTATTCCAGAGCTTAGGTGAACTGTTTATTAGCGCCCTTGGCCTGGCGATGATTGCTGCCCTGGTGCCGCAGCATTTGATGGGCTTTATTCTCGGGATGTGGTTCCTGACGCAGGCTGCCGCGTTCTTGCTGGGCGGCTATGTGGCAACATTTACCGCGGTGCCGGACAACATTACCGATCCGCTTGAGACGTTGCCCGTCTATACCAACGTGTTTGGTAAGATTGGTCTGGTCACGCTGGGCGTTGCAGTAGTGATGCTGTTGATGGTGCCGTGGCTGAAACGCATGATTGCGACGCCGGAAAGCCATTAA。
TABLE 1 OD of original strain 600 Value of
(2) Back-mutating dtpA and dtpB on the basis of high-yielding strains
Based on the high-yield fungus XY-Wa-F1 obtained in the step (1), the original gene fragment is transformed back through RED-XER to replace the original mutant fragment, and the principle is shown in figure 2.
Construction of the mutation cassette fragment: the corresponding primers and the PUC19 plasmid are used as templates to amplify the PUC19 fragment, the genome DNA is used as a template to amplify the upstream and downstream homology arms, and the pMD19-difGm plasmid is used as a template to amplify dif-Gm-up and Gm-dif-down. Five fragments were obtained by gel recovery or purification kit purification. Wherein the upstream primer of the dtpA mutant box does not relate to the coding gene, and the upstream primer of the dtpB mutant box is a pseudogene locus. The primer sequences involved are shown in Table 2.
TABLE 2 names and specific sequences of primers required for construction of mutation cassette fragments
Ligation and transformation:
the system was incubated with Gibson Assembly Master Mix enzyme, 10uL, at 50deg.C for 1h. TOP10 or DH 5. Alpha. Competent cells were transformed. Plates containing Amp (50. Mu.g/mL) and Gm (25. Mu.g/mL) were applied and incubated overnight at 37 ℃. Monoclonal colonies were picked and spotted on primary resistance plates to exclude false positives. The strain after spot shaking, generally about 8-12, is PCR verified by using vector primer M13 +/-to verify that the correct strain selection part is sent for sequencing.
Amplification mutation cassette: the correct strain extract plasmid was sequenced and then the recombinant plasmid containing the mutant cassette fragment was digested and linearized (avoiding the use of restriction sites on the mutant cassette fragment). And (5) recycling the gel to remove the non-target fragments.
Obtaining high-concentration mutation boxes: designing a mutation box primer, and carrying out PCR (polymerase chain reaction) by taking a product obtained by amplifying the mutation box as a template, and purifying the PCR product to obtain a high-concentration mutation box fragment. The mutation cassette primers are shown in Table 3.
TABLE 3 mutation cassette primer sequence information
Preparation of electric conversion competence:
(1) The pKD46 plasmid was transformed into E.coli competence by electrotransformation or chemotransformation to obtain a transformed strain.
(2) The E.coli host of (1) was inoculated on a corresponding antibiotic LB plate (Amp: 50. Mu.g/mL), and cultured at 30℃for 24 hours.
(3) Single colonies were picked and inoculated into 50mL LB (antibiotic working concentration as above) liquid medium (in 250mL Erlenmeyer flasks), cultured at 30℃and 200rpm overnight.
(4) 0.5mL-0.7mL of the culture broth was inoculated into 50mL of fresh LB liquid medium (in 500mL Erlenmeyer flask) containing ampicillin (100. Mu.g/mL) and 10mM L-arabinose, and cultured at 30℃and 220rpm until the A600 value was about 0.7 (about 3-4 hours).
(5) The cells were collected by centrifugation at 4000rpm at 4℃for 5 min.
(6) The medium was poured and the bacteria gently resuspended in 20mL of pre-chilled 10% (v/v) glycerol solution.
(7) The cells were collected by centrifugation and resuspended in 20mL of pre-chilled 10% (v/v) glycerol solution.
(8) Step (6) and step (7) were repeated twice, the supernatant was poured, and the cells were resuspended with a residual of about 500. Mu.L of 10% (v/v) (v/v) glycerol solution.
Electrotransformation competent cells:
(1) 70. Mu.L of the bacterial solution was gently mixed with 15. Mu.L of purified mutant cassette PCR product (100 ng/uL to 150 ng/uL), placed in a 0.2cm cold electric rotating cup and shocked with a shock meter at 2.5kV for an ideal shock time of 4-5ms.
(2) 1mL of LB liquid medium (room temperature) containing 1mM L-arabinose was rapidly added, and the bacterial solution was cultured at 30℃for 3 hours.
(3) The recombinant strain was selected by plating the cells onto LB solid plates containing Gm 10. Mu.g/mL.
Screening and verification of positive transformants:
(1) Colonies growing on the plates of step (3) in electrotransformed competent cells were subjected to one-time spotting on the same resistant plates and colonies growing on new plates were selected for shake verification.
(2) Primers are designed at the upstream and downstream of the gene, bacterial liquid is taken for PCR verification, and the fragment size is verified to be correct, and the fragment can be selectively sent for sequencing or sequenced after the resistance is lost in the next step.
Xer recombinase-mediated removal of Gm resistance gene:
(1) The positive transformants were inoculated in LB liquid medium, cultured at 37℃for 12 hours, and then inoculated with fresh LB liquid medium for further culture for 12 hours.
(2) The culture solution was diluted by 5 passages and three sections were streaked on LB plates and cultured at 37 ℃.
(3) The plates were used to screen gentamicin sensitive transformants (cells that had undergone Xer recombination at the dif locus lost gentamicin resistance), PCR verified with primers and the PCR products sequenced to determine resistant gene knockouts.
Sequencing results Gm resistance deleted.
The obtained strains were designated as F1-R-dtpA and F1-R-dtpB, respectively. The sequences involved are shown in Table 4.
TABLE 4 sequence information
The shake flask fermentation results are shown in Table 5:
TABLE 5 shaking flask fermentation results for each strain
As can be seen from Table 5, the acid production of the back-mutated strains F1-R-dtpA and F1-R-dtpB was reduced compared to XY-Wa-F1, but was still better than that of the original strains.
Example 2: application of mutant in production of L-tryptophan
According to the construction method of the point mutation recombinant strain, construction of the recombinant plasmid: firstly, the dtpA gene in the genome of the tryptophan-producing strain is knocked out, a homologous recombination system is utilized in the experiment, a pKOV plasmid is adopted for the experiment at the stage, the plasmid contains a sucrose lethal gene (sacB) and a temperature sensitive gene (rep 101), the optimum culture temperature is 30 ℃, and the construction method of the pKOV recombinant plasmid is as follows:
the pKOV plasmid was first double digested with NotI/BamHI to obtain linearized plasmid fragments. And then, taking the genome of the tryptophan-producing strain as a template, and respectively taking dtpA1-U-F/dtpA1-U-R and dtpA1-D-F/dtpA1-D-R as primers to amplify to obtain an upstream homology arm and a downstream homology arm of the dtpA, wherein the 5 'end of the upstream homology arm and the 3' end of the downstream homology arm are respectively introduced into a section of homology sequences at two ends (NotI and BamHI) of the linearization plasmid through the primers for efficient connection with the linearization plasmid fragments. The pMAD plasmid genome is used as a template, amp fragments are amplified by using amp-F/amp-R primers, homologous sequences at the 3 'end and the 5' end of an upstream homology arm and a downstream homology arm are respectively contained at two ends of the amp fragments, then the upstream homology arm, the downstream homology arm, the amp fragments and the linearization plasmid fragments are connected by using a recombination kit, the mixture is transformed into E.coli DH5 alpha competent cells by heat shock, the mixture is cultured overnight at 30 ℃, the plasmid genome is extracted from a single colony which is grown, PCR and sequencing verification are carried out by using Vet-F/R, and the verified recombinant plasmid is named as pKOV-HR. dtpB was constructed in the same manner and the primers are shown in Table 6.
TABLE 6 primer information
Obtaining a strain containing a marker gene: the recombinant plasmid pKOV-HR constructed above was transformed into competent cells of E.coli, spread on plates containing ampicillin and chloramphenicol resistance (hereinafter abbreviated as "diabody") and cultured overnight at 30 ℃. The single colony is randomly picked and coated on a double-antibody flat plate, and is cultured at 42 ℃ overnight, and the plasmid does not contain a homologous repair arm and cannot normally replicate in a host under the high-temperature culture condition because the plasmid has a temperature-sensitive gene rep101, and the recombinant plasmid containing the homologous repair arm can undergo homologous recombination with the host genome, so that plasmid DNA is integrated into the host genome. Single colonies were randomly picked from 42 ℃ culture plates and plated onto ampicillin resistant plates containing 5% sucrose, incubated overnight at 30 ℃ for secondary homologous recombination to effect replacement of the amp marker gene with the target gene, while pKOV plasmid was eliminated. The strains with recombination are screened by adopting a 96-well plate, the same single colony is selected and respectively inoculated into LB culture solution with ampicillin resistance and chloramphenicol resistance, and the strain is placed in a shaking table at 30 ℃ for overnight culture at 150 r/min. Colonies growing in ampicillin resistant medium but not in chloramphenicol resistant medium were selected, grown up and genome verified. Specific fragment amplification was performed using Vet-F/R and submitted to sequencing company for sequencing and further result verification to obtain target gene knockouts while ampicillin resistant E.coli ΔdtpA:: ampR or ΔdtpB::: ampR.
E.coli target gene point mutation experiment:
the point mutation experiment of the research is carried out on the basis of the strain with the ampicillin gene obtained in the previous step, and adopts a CRISPR/Cas9 double-plasmid system to realize the point mutation of a target gene, wherein pSGKP-km has a sgRNA sequence and a sucrose lethal gene (sacB), and is simultaneously used as a carrier plasmid for connecting a spacer and a homologous repair arm; the pCasKP-apr plasmid was used to express Cas9 protein and lambda-Red recombinase, with a temperature sensitive gene (rep 101). Construction of recombinant plasmids: the tryptophan producing strain DeltadtpA obtained by the one-step knockout experiment is characterized in that ampR or DeltadtpB is characterized in that an ampR genome is used as a template, a space sequence which can identify 20-nt of an amp gene is shown as SEQ ID NO.67 (5'-AAAAGGGAATAAGGGCGACA-3') in http:// www.rgenome.net/design, 4 bases (5 '-TAGT-) which are homologous to BsaI cleavage sites of a pSGKP-km plasmid are added to the 5' -end of the space sequence for connecting the space with the pSGKP-km plasmid, then the space sequence is phosphorylated, and the phosphorylated space is connected with a linearized (BsaI) pSGKP-km plasmid by using T4DNA ligase to obtain the pSGKP-km-space plasmid. The fragment of dtpA (A430C) or dtpB (T116) G containing the point mutation is obtained by adopting an overlap extension PCR method, and both ends of the fragment are provided with an upstream homologous repair arm and a downstream homologous repair arm of the gene. The homologous repair arm was ligated with pSGKP-km-spacer plasmid linearized with BamH I, transformed into E.coli DH 5. Alpha. Competent cells by heat shock, plated on kanamycin resistance plates, cultured overnight at 30℃to obtain single colonies, PCR-verified and sequencing-verified using Vet-F/R, and the recombinant plasmid after verification was named pSGKP-km-spacer-HR.
Acquisition of the Point mutant Strain: first, E.coli competent cells were prepared which had been transformed with pCasKP-apr plasmid into tryptophan producer strain ΔdtpA::: ampR or ΔdtpB:::: ampR cells, and during the preparation of competent cells, the expression of Cas9 protein and lambda-Red recombinase was induced by arabinose addition. pSGKP-km-spacer-HR recombinant plasmid was transformed into competent cells described above, plated onto arabinose-added kanamycin and apramycin double antibody plates, and incubated overnight at 30 ℃. Single colonies were picked and plated onto apramycin resistant plates containing 5% sucrose and incubated overnight at 30 ℃. And (3) using a 96-well plate, picking the same single colony, respectively inoculating the single colony into three LB culture solutions without resistance, ampicillin resistance and kanamycin resistance, screening colonies which do not grow in ampicillin and kanamycin antibiotic culture solutions, extracting genome, carrying out PCR and sequencing verification, wherein a sequencing primer is dtpA3-F/R (dtpB 3-F/R), and the verification result is correct, namely the escherichia coli point mutation strains XY-dtpA-pm and XY-dtpB-pm. The pCasKP-apr plasmid was removed by culturing overnight at 42 ℃. The experimental procedure is shown in figure 3.
The XY-dtpAB-pm construction method is that the point mutation dtpB is obtained on the basis of the XY-dtpA-pm.
The shake flask fermentation results are shown in Table 7.
TABLE 7 shaking flask fermentation results of mutant strains
As shown in Table 7, the productivity of the strains XY-dtpA-pm and XY-dtpB-pm were both improved, and the productivity of the double point mutant XY-dtpAB-pm strain was more significantly improved.
Construction of a 5-copy number plasmid overexpression vector for point mutation (three vectors: single gene two, double gene).
The recombinant strain with three over-expressed plasmids is obtained, the single genes (dtpA and dtpB) are over-expressed, the double genes (dtpA and dtpB) are over-expressed and fermented, the yield is obviously improved compared with the yield of the point mutation (third) and the yield of the double-gene plasmid over-expressed strain is obviously improved.
The dtpA and dtpB were amplified using XY-Wa-F1 genomic DNA as templates, and the primers are shown in Table 8.
TABLE 8 amplification of primer sequences involved in dtpA and dtpB Using XY-Wa-F1 genomic DNA as templates
dtpA-F and dtpA-R are used for amplifying and constructing pTH18KR-PJ23119-Ptrc-PamyL-dtpA low-copy plasmid overexpression vector;
dtpB-F, dtpB-R is used for amplifying and constructing pTH18KR-PJ23119-Ptrc-PamyL-dtpB low-copy plasmid overexpression vector; primers dtpA-F and dtpAB-l-R; primers dtpAB-l-F and dtpB-R post amplification two fragments were used to construct
pTH18KR-PJ23119-Ptrc-PamyL-dtpA-dtpB low copy plasmid overexpression vector.
A schematic representation of the pTH18KR-PJ23119-Ptrc-PamyL-T7 terminator plasmid is shown in FIG. 4.
pTH18KR-PJ23119-Ptrc-PamyL-T7 terminator plasmid is extracted, digested with EcoRI and XbaI, and connected with amplified fragment by commercial homologous recombinase to transform commercial E.coli competence.
After the single colony in the last step is identified by PCR, the single colony is amplified and cultured, plasmids are extracted, and part of the plasmid is taken and sent to sequencing. The sequencing primers were as follows:
sequencing-F (SEQ ID NO. 67): 5'-atgaccatgattacgccgagct-3'
sequencing-R (SEQ ID NO. 68): 5'-attaagttgggtaacgccagggttttc-3'
After the sequencing result is completely correct, the plasmid is introduced into escherichia coli competence prepared by tryptophan stock.
The PCR identification is carried out to obtain pTH18KR-PJ23119-Ptrc-Pamy L-dtpA (144N > H) point mutant plasmid overexpression strain XY-O-dtpA-p, pTH18KR-PJ23119-Ptrc-Pamy L-dtpB (39V > G) point mutant plasmid overexpression strain XY-O-dtpB-p and pTH18KR-PJ23119-Ptrc-Pamy L-dtpA (144N > H) -dtpB (39V > G) double point mutant plasmid overexpression strain XY-O-dtpAB-p.
The shake flask fermentation results are shown in Table 9:
TABLE 9 shaking flask fermentation results
As shown in Table 9, the productivity of the strains XY-O-dtpA-p and XY-O-dtpB-p were both improved, and the productivity of the XY-O-dtpAB-p strain was more significantly improved.

Claims (9)

1. A double-peptide tripeptide permease mutant dtpA for improving the yield of L-tryptophan is characterized in that the amino acid sequence of the double-peptide tripeptide permease mutant dtpA is shown in SEQ ID NO. 1.
2. A coding gene of the dipeptide tripeptide permease mutant dtpA according to claim 1, wherein the coding gene is shown in SEQ ID No. 3.
3. A recombinant vector comprising the coding gene of claim 2.
4. A recombinant bacterium comprising the recombinant vector according to claim 3.
5. Use of the dipeptide tripeptide permease mutant dtpA according to claim 1 for the production of L-tryptophan.
6. Use of the coding gene according to claim 2 for the production of L-tryptophan.
7. Use of the recombinant vector of claim 3 for the production of L-tryptophan.
8. The use of the recombinant bacterium of claim 4 for producing L-tryptophan.
9. A method for increasing the yield of L-tryptophan, characterized in that the method is to express mutant dtpA in escherichia coli, or to express both mutant dtpA and mutant dtpB; the coding gene of the mutant dtpA is shown as SEQ ID NO. 3; the coding gene of the mutant dtpB is shown as SEQ ID NO. 4.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101115832A (en) * 2004-11-26 2008-01-30 协和发酵工业株式会社 Industrially useful microorganism

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101115832A (en) * 2004-11-26 2008-01-30 协和发酵工业株式会社 Industrially useful microorganism

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* Cited by examiner, † Cited by third party
Title
"dipeptide and tripeptide permease B [Escherichia coli 2866450]";匿名;《GenBank》;CDS和ORIGIN部分 *
"Knocking out analysis of tryptophan permeases in Escherichia coli for improving L-tryptophan production";Pengfei Gu et al.,;《Appl Microbiol Biotechnol》;第97卷;第6677-6683页尤其是摘要 *
匿名."dipeptide and tripeptide permease B [Escherichia coli 2866450]".《GenBank》.2013,CDS和ORIGIN部分. *

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