L-histidine production strain and construction method and application thereof

Abstract

The application provides an L-histidine producing strain and a construction method and application thereof, and the strain is constructed by using E. coli W3110 as a chassis strain, deleting hisLG, ushA, surE, nagD, nrdD genes, and heterologously expressing hisG*, zwf*, gnd* genes from ATCC 13032 by using a trc promoter, and simultaneously overexpressing hisDCBHAFI, prsA*, an artificial operon rpiAB gene by using a trc promoter. Corynebacterium glutamicum The strain can effectively improve the L-histidine synthesis efficiency by relieving the feedback inhibition of key enzymes, knocking out the branch pathway gene of ATP synthesis, strengthening the expression of the key enzymes in the pathway, and improving the PRPP content, is genetically stable, does not need to add resistant substances, and has good L-histidine synthesis capacity and high acid production efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology and fermentation engineering, and particularly relates to an L-histidine production strain and a construction method and application thereof. BACKGROUND

[0002] Histidine is one of 20 standard protein amino acids and participates in protein synthesis. L-histidine can be widely used in various fields of biological medicine and has various physiological functions and is widely used in various fields of food, medicine and the like.

[0003] At present, main methods for preparing L-histidine can be divided into protein hydrolysis, chemical synthesis and microbial fermentation. Although the protein hydrolysis method is mature, the method has great limitations in application and development due to serious corrosion of equipment, high loss rate of hydrolysis, low yield and serious environmental pollution. The chemical synthesis method has complex production steps and long cycle, and the obtained product is mostly DL racemate. Compared with the protein hydrolysis method and the chemical synthesis method, the microbial fermentation method has mild conditions, small environmental pollution, easy process control and cheap fermentation raw materials, and is the preferred method for future industrial production of histidine.

[0004] In recent years, although the microbial fermentation develops rapidly, there are still problems of low yield, low sugar acid conversion rate, high production cost and long fermentation cycle in the production of histidine. Therefore, it is a technical problem to be solved at present to construct an L-histidine production strain with high acid production efficiency and good genetic stability. SUMMARY

[0005] The present application provides an L-histidine production strain.

[0006] Another technical problem to be solved by the present application is to provide a construction method of the L-histidine production strain.

[0007] Another technical problem to be solved by the present application is to provide an application of the L-histidine production strain.

[0008] To solve the above technical problems, the technical scheme of the present application is as follows:

[0009] An artificial operon is named as artificial operon rpiAB, and the nucleotide sequence is shown in the sequence table SEQ ID NO. 16.

[0010] Preferably, the artificial operon comprises an rpiA gene and an rpiB gene, and specifically comprises:

[0011] (1) the gene order of the operon is trc promoter, rpiA, RBS1, rpiB, rrnB T1 terminator;

[0012] (2) the trc promoter is directly connected with the rpiA gene at the head and tail;

[0013] (3) the rpiA and rpiB are connected through the RBS1 gene;

[0014] (4) the rpiB and the rrnB T1 terminator are directly connected at the head and tail.

[0015] Preferably, the nucleotide sequence of the rpiA gene is shown in the sequence table SEQ ID NO. 11, the nucleotide sequence of the rpiB gene is shown in the sequence table SEQ ID NO. 12, the nucleotide sequence of the RBS1 gene is shown in the sequence table SEQ ID NO. 14, and the nucleotide sequence of the rrnB T1 terminator is shown in the sequence table SEQ ID NO. 15.

[0016] The construction method of the artificial operon is that the transcription of the operon is controlled by the same trc promoter, the transcription of the operon is controlled by the same rrnB T1 terminator, and the gene connection mode and the position of the gene in the operon are optimized.

[0017] An L-histidine producing strain, named strain YTH13, has all the characteristics of the artificial operon described above, and is obtained by the following method. E. coli W3110 as the chassis strain, the hisLG, ushA, surE, nagD, nrdD genes are deleted, the trc promoter is used to express the hisG*, zwf*, gnd* genes from ATCC 13032 heterologously, and the trc promoter is used to overexpress the hisDCBHAFI, prsA*, artificial operon rpiAB genes. Corynebacterium glutamicum ATCC 13032, and the trc promoter is used to overexpress the hisDCBHAFI, prsA*, artificial operon rpiAB genes.

[0018] Preferably, the L-histidine producing strain is obtained by the following method. E. coli W3110 as the chassis strain, the hisLG, ushA, surE, nagD, nrdD genes are deleted, the trc promoter is used to express the hisG*, zwf*, gnd* genes from ATCC 13032 heterologously, and the trc promoter is used to overexpress the hisDCBHAFI, prsA*, artificial operon rpiAB genes. Corynebacterium glutamicum ATCC 13032, and the trc promoter is used to overexpress the hisDCBHAFI, prsA*, artificial operon rpiAB genes. Corynebacterium glutamicum ATCC 13032, and the trc promoter is used to overexpress the hisDCBHAFI, prsA*, artificial operon rpiAB genes. Corynebacterium glutamicumthe gnd* gene transcription and integrated into the genome ygaY pseudogene site; the prsA* gene transcription was enhanced by trc promoter and integrated into the genome yghE pseudogene site; the hisDCBHAFI gene transcription was enhanced by trc promoter and integrated into the genome ilvG pseudogene site; the rpiAB artificial operon gene transcription was enhanced by trc promoter and integrated into the genome ycgH pseudogene site.

[0019] Preferably, the L-histidine production strain has the nucleotide sequence of the hisLG gene as shown in the sequence listing SEQ ID NO. 1, the nucleotide sequence of the ushA gene as shown in the sequence listing SEQ ID NO. 2, the nucleotide sequence of the surE gene as shown in the sequence listing SEQ ID NO. 3, the nucleotide sequence of the nagD gene as shown in the sequence listing SEQ ID NO. 4, and the nucleotide sequence of the nrdD gene as shown in the sequence listing SEQ ID NO. 5.

[0020] Preferably, the L-histidine production strain has the nucleotide sequence of the hisG* gene as shown in the sequence listing SEQ ID NO. 6, the nucleotide sequence of the zwf* gene as shown in the sequence listing SEQ ID NO. 7, the nucleotide sequence of the gnd* gene as shown in the sequence listing SEQ ID NO. 8, the nucleotide sequence of the prsA* gene as shown in the sequence listing SEQ ID NO. 9, the nucleotide sequence of the hisDCBHAFI gene as shown in the sequence listing SEQ ID NO. 10, and the nucleotide sequence of the trc promoter as shown in the sequence listing SEQ ID NO. 13.

[0021] Preferably, the L-histidine production strain has the nucleotide sequence of the hisG* gene as shown in the sequence listing SEQ ID NO. 6, the nucleotide sequence of the zwf* gene as shown in the sequence listing SEQ ID NO. 7, the nucleotide sequence of the gnd* gene as shown in the sequence listing SEQ ID NO. 8, the nucleotide sequence of the prsA* gene as shown in the sequence listing SEQ ID NO. 9, the nucleotide sequence of the hisDCBHAFI gene as shown in the sequence listing SEQ ID NO. 10, and the nucleotide sequence of the trc promoter as shown in the sequence listing SEQ ID NO. 13. E. coli W3110 is E. coli W3110 ATCC 27325.

[0022] The method for constructing the L-histidine production strain is performed on the starting strain W3110 by targeted modification, and the specific steps are as follows: E. coli The method for constructing the L-histidine production strain is performed on the starting strain W3110 by targeted modification, and the specific steps are as follows:

[0023] (1) chassis modification: relieve L-histidine feedback regulation: knock out hisLG; introduce the hisG* gene from E. coli K-12 MG1655; Corynebacterium glutamicum

[0024] (2) strengthen the expression of key enzymes in the pathway: overexpress hisDCBHAFI using trc promoter, so that the key enzymes in the L-histidine metabolic pathway are highly expressed, thereby greatly improving the yield of L-histidine;

[0025] ​(3) Strengthen the synthesis of precursor PRPP: prsA*, artificial operon rpiAB genes are overexpressed using trc promoter; zwf*, gnd* genes derived from Corynebacterium glutamicum are introduced to increase the supply of precursor substances to facilitate the efficient production of end products;

[0026] (4) Strengthen the synthesis of ATP: knock out ushA, surE, nagD, nrdD genes to enhance energy supply, which is conducive to the efficient production of end products.

[0027] The application of the above-mentioned L-histidine production strain in the fermentation production of L-histidine.

[0028] The application of the above-mentioned L-histidine production strain is to ferment L-histidine in the culture medium using the L-histidine production strain under suitable fermentation conditions, which includes but is not limited to carbon source, nitrogen source, inorganic salt, vitamin, etc.; fermentation conditions include fermentation temperature, fermentation pH, fermentation dissolved oxygen condition, fermentation pressure, fermentation time, etc. The culture medium can be obtained by conventional methods and used for L-histidine production, and the fermentation conditions can be adjusted to adapt to the production characteristics of the strain.

[0029] Preferably, the application of the above-mentioned L-histidine production strain has the following specific steps:

[0030] ① Inclined surface culture: inoculate the L-histidine production strain on the inclined surface culture medium and culture;

[0031] ② Seed culture in a shake flask: inoculate the solid inclined surface strain into the shake flask culture medium for fermentation;

[0032] ③ Shake flask fermentation culture: the fermentation inoculation amount is 20%-25%.

[0033] Preferably, the application of the above-mentioned L-histidine production strain has the following specific steps:

[0034] ① Inclined surface culture: inoculate the L-histidine production strain on the inclined surface culture medium and culture at 37℃ for 12h;

[0035] ② Seed culture in a shake flask: inoculate the solid inclined surface strain into the shake flask culture medium for fermentation, the culture temperature is 37℃, the culture time is 12h, the shaking bed rotation speed is 220r / min, and the pH is 7.0;

[0036] ③ Shake flask fermentation culture: the fermentation inoculation amount is 20%-25%, the culture temperature is 37℃, the pH is 7.0, the culture time is 24h, and the shaking bed rotation speed is 220r / min.

[0037] Preferably, the application of the above-mentioned L-histidine production strain uses the general LB solid culture medium in step ①.

[0038] Preferably, in the application of the above L-histidine producing strain, the seed culture medium used in step ② is: glucose 30 g / L, yeast powder 6 g / L, proteose peptone 2 g / L, MgSO4·7H2O 0.8 g / L, KH2PO4·3H2O 2 g / L, ammonium sulfate 1 g / L, FeSO4·7H2O 10 mg / L, threonine 1 g / L, methionine 0.5 g / L, citric acid 2 g / L, and the rest is water.

[0039] Preferably, in the application of the above L-histidine producing strain, the fermentation medium used in step ③ is: MgSO4·7H2O 1.5 g / L, yeast powder 5 g / L, proteose peptone 2 g / L, ammonium sulfate 3 g / L, methionine 0.2 g / L, K2HPO4·3H2O 4 g / L, citric acid 2.5 g / L, threonine 1 g / L, lysine 1 g / L, isoleucine 0.5 g / L, FeSO4·7H2O 20 mg / L, MnSO4 20 mg / L, MgSO4·7H2O 1.5 g / L, VB1, VB3, VB5 each 2 mg / L, and the rest is water.

[0040] The above culture media can be prepared by standard methods.

[0041] Beneficial effects:

[0042] The above L-histidine producing strain does not contain plasmids, is genetically stable, does not need to add resistance substances, has good L-histidine synthesis capacity, low production cost, stable performance, high acid production efficiency, and good economic benefits; the strain can effectively improve the synthesis efficiency of L-histidine and the production level of L-histidine by relieving the feedback inhibition of key enzymes, knocking out the branch pathway genes of ATP synthesis, strengthening the expression of key enzymes in the pathway, and increasing the content of PRPP, realizing the efficient production of L-histidine, and having excellent industrial application prospect. Specifically:

[0043] (1) By knocking out hisL, the inhibition of histidine structural gene transcription is relieved; by knocking out hisG and introducing the hisG* gene from Escherichia coli, the feedback inhibition of histidine is relieved, and by using the trc promoter to overexpress hisDCBHAFI, the efficient expression of key enzymes in the L-histidine metabolic pathway is realized, thereby greatly improving the yield of L-histidine; Corynebacterium glutamicum

[0044] (2) By knocking out ushA, surE, nagD, and nrdD genes, the metabolism of ATP synthesis branch pathway is reduced, the energy supply is enhanced, and the efficient production of end products is facilitated;

[0045] (3) By using the trc promoter to overexpress prsA* and the artificial operon rpiAB genes, and by introducing the hisG* gene from Escherichia coli, the feedback inhibition of histidine is relieved, and the efficient expression of key enzymes in the L-histidine metabolic pathway is realized, thereby greatly improving the yield of L-histidine;​Corynebacterium glutamicum The zwf* and gnd* genes increase the synthesis of the precursor PRPP, thereby greatly increasing the production of L-histidine. Attached Figure Description

[0046] Figure 1 A diagram illustrating the de novo synthesis pathway gene modification process for L-histidine-producing strains; where GUO: 5-(5-phosphate-D-ribosecarbamoyl)-1-(5-phosphateribosyl)-imidazol-4-carboxamide; 2ER: 5-[(5-phosphate-1-deoxyribone-1-amino)methylacetamide]-1-(5-phosphateribosyl)imidazol-4-carboxamide. Detailed Implementation

[0047] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0048] Unless otherwise specified, the percentage sign "%" used in the examples refers to the 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.

[0049] The starting strain used in the examples was wild-type. E. coli W3110 ATCC 27325 (commercially available), the corresponding promoter and gene are shown in the sequence listing. Primers used in the construction of the involved strains are shown in Table 1.

[0050] Table 1 Primers used in strain construction

[0051] Primer name Sequence No. Primer sequence (5'-3') hisLG-pGRB-S SEQ ID NO. 17 AGTCCTAGGTATAATACTAGTCGGCGGCTGTCGTCTTTCGCGTTTTAGAGCTAGAA hisLG-pGRB-A SEQ ID NO 18 TTCTAGCTCTAAAACGCGAAAGACGACAGCCGCCGACTAGTATTATACCTAGGACT hisLG-U-S SEQ ID NO. 19 GATTTCAGGCTATCGATTGAGTCCATCA hisLG-U-A SEQ ID NO. 20 TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAACCACTTTCACGTTAGAAAGCAAGG hisLGD-S SEQ ID NO. 21 TCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAGCTTTAACACAATCATTGACTGGAAT hisLG-D-A SEQ ID NO. 22 CCAGCATTAATACCGTTGAGAAGAGC SEQ ID NO. 23 AGTCCTAGGTATAATACTAGTGGCTGGATCACCGCAGAGTAGTTTTAGAGCTAGAA SEQ ID NO. 24 TTCTAGCTCTAAAACTACTCTGCGGTGATCCAGCCACTAGTATTATACCTAGGACT SEQ ID NO. 25 ATAGCGCAGGGTACATTCCACT SEQ ID NO. 26 GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCTTCTTCAATAGAGGCGGTACA SEQ ID NO. 27 TGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTGCCGCAGAGACCGACAT SEQ ID NO. 28 ACAGCGGTTGTGGTGGCA SEQ ID NO. 29 TATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTTGAAAATCGCTGTCCCAAAC SEQ ID NO. 30 CACCAAGCTTGATGAATACCTCTAC SEQ ID NO. 31 GTAGAGGTATTCATCAAGCTTGGTG SEQ ID NO. 32 aaacaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttGGAAGTTCTGCGCGTGCAAA SEQ ID NO. 33 AGTCCTAGGTATAATACTAGTTGAACAGTTTACCGGTGCGGGTTTTAGAGCTAGAA SEQ ID NO. 34 TTCTAGCTCTAAAACCCGCACCGGTAAACTGTTCAACTAGTATTATACCTAGGACT SEQ ID NO. 35 GGTCAGGAGGTAACTTATCAGCG SEQ ID NO. 36 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAATGGCAGGGCTCCGTTTTG SEQ ID NO. 37 CCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGAACTGGATTTTCTTCTGAACCTGTCG SEQ ID NO. 38 ACGATGTCAGCAGCCAGC -pGRB-S SEQ ID NO. 39 AGTCCTAGGTATAATACTAGTTATATCGCCCTGGCACCTGAGTTTTAGAGCTAGAA -pGRB-A SEQ ID NO. 40 TTCTAGCTCTAAAACTCAGGTGCCAGGGCGATATAACTAGTATTATACCTAGGACT -U-S SEQ ID NO. 41 TCAAACGCTTTACGCAGGAT -U-A SEQ ID NO. 42 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTGATGGCAACAACAGGGA -D-S SEQ ID NO. 49 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCTATCTACGCGCCGTTGTTGTT -D-A SEQ ID NO. 50 GCGCTGGCTAACATGAGGAA SEQ ID NO. 51 CTGAACAACATCATATTTAAATGAACATAACTCAATTTGTAGGCTAGCATAACCCCTTGGGGCCTATCTACGCGCCGTTGTTGT SEQ ID NO. 52 ATGCACAGGAGACTTTCTGATGCGCTGGTTGATTTCTTCTAGGGTCATAGTAATCCAGCAACTCTATCTACGCGCCGTTGTTGT hisD-S SEQ ID NO. 53 TCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAGCTTTAACACAATCATTGACTGGAAT hisD-A-1# SEQ ID NO. 54 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTGATGGCAACAACAGGGA -D-S SEQ ID NO. 55 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCTATCTACGCGCCGTTGTTGTT -D-A SEQ ID NO. 56 GCGCTGGCTAACATGAGGAA SEQ ID NO. 57 CTGAACAACATCATATTTAAATGAACATAACTCAATTTGTAGGCTAGCATAACCCCTTGGGGCCTATCTACGCGCCGTTGTTGT SEQ ID NO. 58 ATGCACAGGAGACTTTCTGATGCGCTGGTTGATTTCTTCTAGGGTCATAGTAATCCAGCAACTCTATCTACGCGCCGTTGTTGT hisD-S SEQ ID NO. 59 TCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAGCTTTAACACAATCATTGACTGGAAT hisD-A-1# SEQ ID NO. 60 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTGATGGCAACAACAGGGA -D-S SEQ ID NO. 61 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCTATCTACGCGCCGTTGTTGTT -D-A SEQ ID NO. 62 GCGCTGGCTAACATGAGGAA SEQ ID NO. 63 CTGAACAACATCATATTTAAATGAACATAACTCAATTTGTAGGCTAGCATAACCCCTTGGGGCCTATCTACGCGCCGTTGTTGT SEQ ID NO. 64 ATGCACAGGAGACTTTCTGATGCGCTGGTTGATTTCTTCTAGGGTCATAGTAATCCAGCAACTCTATCTACGCGCCGTTGTTGT hisD-S SEQ ID NO. 65 TCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAGCTTTAACACAATCATTGACTGGAAT hisD-A-1# SEQ ID NO. 66 GCCCCAAGGGGTTATGCTAGCCTACAAATTGAGTTATGTTCATTTAAATATGATGTTGTTCAGGCAGTTCAATACCTTCGTCCGC hisCB-S SEQ ID NO. 55 GCTGAACGCCAACGAATACCC hisCB-A-4 SEQ ID NO. 56 AGTTGCTGGATTACTATGACCCTAGAAGAAATCAACCAGCGCATCAGAAAGTCTCCTGTGCATCGATCATTTCGGTGCTGAGATCG hisHA-S SEQ ID NO. 57 CGGTTTCCGCATGGAAATCAAC hisHA-A-1 SEQ ID NO. 58 GCCCCAAGGGGTTATGCTAGCCTACAAATTGAGTTATGTTCATTTAAATATGATGTTGTTCAGGGAGGACTGAAATGCCACCTG hisFI-S SEQ ID NO. 59 GAGAACTCGGGCGTTTCACT hisFI-A SEQ ID NO. 60 caccgacaaacaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttGTCACTGATGCCGTTTACGCAG SEQ ID NO. 61 AGTCCTAGGTATAATACTAGTATGAACATAACTCAATTTGTGTTTTAGAGCTAGAA SEQ ID NO. 62 TTCTAGCTCTAAAACACAAATTGAGTTATGTTCATACTAGTATTATACCTAGGACT SEQ ID NO. 63 AGTCCTAGGTATAATACTAGTTGCGCTGGTTGATTTCTTCTGTTTTAGAGCTAGAA SEQ ID NO. 64 TTCTAGCTCTAAAACAGAAGAAATCAACCAGCGCAACTAGTATTATACCTAGGACT -pGRB-S SEQ ID NO. 65 AGTCCTAGGTATAATACTAGTTATGCGTCTGAACGACCGTGGTTTTAGAGCTAGAA -pGRB-A SEQ ID NO. 66 TTCTAGCTCTAAAACCACGGTCGTTCAGACGCATAACTAGTATTATACCTAGGACT -U-S SEQ ID NO. 67 TAAACTCGTCAGCGGCACAAC -U-A SEQ ID NO. 68 GATTCAGGTTGCCATTTACGC SEQ ID NO.71 CGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGACGCAGGATGAATTGAAAAAAGCA SEQ ID NO.72 TCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGACTCATTTCACAATGGTTTTGACACCGT SEQ ID NO.73 TTTGTTTAACTTTAAGAAGGAGATATACCATGAAAAAGATTGCATTTGGCTGTGATCA SEQ ID NO.74 gacaaacaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttGTCAATTTCTCCGCTGCTCTATTGCC -pGRB-S SEQ ID NO.75 AGTCCTAGGTATAATACTAGTAACACAGCAATACGGTACGCGTTTTAGAGCTAGAA -pGRB-A SEQ ID NO.76 TTCTAGCTCTAAAACGCGTACCGTATTGCTGTGTTACTAGTATTATACCTAGGACT -U-S SEQ ID NO.77 TTCATCGGGACGAGTGGAGA -U-A SEQ ID NO.78 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCATAGCATCGCCAATCTGA -D-S SEQ ID NO.79 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACCCAAAGGTGAAGATAAAGCC -D-A SEQ ID NO.80 CATTCCCTCTACAGAACTAGCCCT SEQ ID NO.81 ​ ​ ​ ​ ​ -1-S ​ TGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCgtgagcacaaacacgacccc -1-A SEQ ID NO. 82 acccaagccaatatcttcagtcatg -2-S SEQ ID NO. 83 catgactgaagatattggcttgggt -2-A SEQ ID NO. 84 caacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttGttatggcctgcgccaggt -pGRB-S SEQ ID NO. 85 AGTCCTAGGTATAATACTAGTCACTGATGGCGCTGGCATTAGTTTTAGAGCTAGAA -pGRB-A SEQ ID NO. 86 TTCTAGCTCTAAAACTAATGCCAGCGCCATCAGTGACTAGTATTATACCTAGGACT -U-S SEQ ID NO. 87 CCTACAAACCACATCGCACATT -U-A SEQ ID NO. 88 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAACACCGAAGCAACCCAAAAG -D-S SEQ ID NO. 89 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTGCTTGCCGCTCCACC -D-A SEQ ID NO. 90 GGAGTAGGGCTTTCCATAGAGTGT -1-S SEQ ID NO. 91 TGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCatgccgtcaagtacgatcaataacatg -1-A SEQ ID NO. 92 ttctcgtcgaagccagcctt -2-S SEQ ID NO. 93 aaggctggcttcgacgagaa -2-A SEQ ID NO. 94 cagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttGttaagcttcaacctcggagcg SEQ ID NO. 95 AGTCCTAGGTATAATACTAGTGCTGAAAAAATATCGCCCACGTTTTAGAGCTAGAA SEQ ID NO. 96 TTCTAGCTCTAAAACGTGGGCGATATTTTTTCAGCACTAGTATTATACCTAGGACT SEQ ID NO. 97 GTCAGGCACTGGCGAAAGAT SEQ ID NO. 98 TTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACGCAAGCCATAAACCCACAAG SEQ ID NO. 99 GCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTCCGACATCGAAATGCGTGG SEQ ID NO. 100 AGGCGTTGTTGTGGCAGAT SEQ ID NO. 101 GCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCgtgcctgatatgaagctttttgctgg SEQ ID NO. 102 cgtgcagagccactgtcag SEQ ID NO. 103 ctgacagtggctctgcacg SEQ ID NO. 104 acaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttGttagtgttcgaacatggcagagatcg SEQ ID NO. 105 AGTCCTAGGTATAATACTAGTaaacaggtcgattacgtgccGTTTTAGAGCTAGAA SEQ ID NO. 106 TTCTAGCTCTAAAACggcacgtaatcgacctgtttACTAGTATTATACCTAGGACT SEQ ID NO. 107 aaattattgcagcggggcgt SEQ ID NO. 108 tatcgcctgcttcgatagaatcacctggtagatattcgcggaaagca SEQ ID NO. 109 tgctttccgcgaatatctaccaggtgattctatcgaagcaggcgata SEQ ID NO. 110 tactgccagctcacctcacc SEQ ID NO. 111 AGTCCTAGGTATAATACTAGTCGCCCGGACATTGTTGTGTCGTTTTAGAGCTAGAA SEQ ID NO. 112 TTCTAGCTCTAAAACGACACAACAATGTCCGGGCGACTAGTATTATACCTAGGACT SEQ ID NO. 113 TGGCATTCGAACAAGCAGCT SEQ ID NO. 114 GCGTCACGCGAATACCTTTGGTCAGAGAATTTGAAGCGCCG SEQ ID NO. 115 CGGCGCTTCAAATTCTCTGACCAAAGGTATTCGCGTGACGC SEQ ID NO. 116 GTCAGCTCGAGTAATTCGGTCA SEQ ID NO. 117 AGTCCTAGGTATAATACTAGTCACGATAACGTCGCCGTACCGTTTTAGAGCTAGAA SEQ ID NO. 118 TTCTAGCTCTAAAACGGTACGGCGACGTTATCGTGACTAGTATTATACCTAGGACT SEQ ID NO. 119 AAACCATCGCCATTGCTATCAAC SEQ ID NO. 120 CATTTTGTTTAATGCTGCGCGGAAATGGTCATGGACTACCCAGAATATTG SEQ ID NO. 121 CAATATTCTGGGTAGTCCATGACCATTTCCGCGCAGCATTAAACAAAATG SEQ ID NO. 122 GGGGCCAAATTGCTGACAAAGTG SEQ ID NO. 123 AGTCCTAGGTATAATACTAGTGAACCTGCCGCGTATTGCTCGTTTTAGAGCTAGAA SEQ ID NO. 124 TTCTAGCTCTAAAACGAGCAATACGCGGCAGGTTCACTAGTATTATACCTAGGACT SEQ ID NO. 125 TTGCTACTACAGCTCCCCACGAAAAA SEQ ID NO. 126 TCAATCGGTGTATTGGTGCCGTAATCGATCGTGACGGTACTCGATGT SEQ ID NO. 127 ACATCGAGTACCGTCACGATCGATTACGGCACCAATACACCGATTGA SEQ ID NO. 128 TGACGATGTCGACAGGATAGTACTG

[0052] like Figure 1 As shown, the strain was modified according to the metabolic pathway of the entire strain modification process to obtain L-histidine producing strain YTH13. The modification process mainly includes the following four modules:

[0053] (1) Chassis bacteria modification: Disruption of L-histidine feedback regulation: Knockout of hisLG; Introduction of bacteria derived from Corynebacterium glutamicum the hisG* gene;

[0054] (2) Enhance the expression of key enzymes in the pathway: hisDCBHAFI was overexpressed using the trc promoter, which enabled the efficient expression of key enzymes in the L-histidine metabolic pathway, thereby greatly increasing the yield of L-histidine.

[0055] (3) Enhance the synthesis of the precursor PRPP: The prsA* gene and the artificial operon rpiAB gene were overexpressed using the trc promoter; and a gene derived from... Corynebacterium glutamicumThe zwf* and gnd* genes, increasing the supply of precursor substances, are beneficial to the efficient production of the final product;

[0056] (4) Enhance ATP synthesis: Knockout of ushA, surE, nagD and nrdD genes enhances energy supply and is conducive to the efficient production of end products.

[0057] The gene editing methods used in the above gene manipulations refer to 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 specified, all technical terms used in this invention are explained within the context of this article. In this invention, "knockout" refers to the inactivation of the target gene, and "introduction" refers to the insertion of a foreign gene into the genome of an engineered bacterium after linking it with a promoter and terminator.

[0058] Example 1

[0059] This embodiment aims to illustrate the specific construction steps of strain YTH13. In particular, if there are similar gene manipulation methods in the embodiment, they will only be provided once and annotated, without further elaboration.

[0060] The specific steps for constructing the L-histidine-producing strain are as follows:

[0061] (1) Knockout of the hisLG gene: E. coli Using the W3110 genome as a template, upstream and downstream homologous arms were amplified by PCR using hisLG-US, hisLG-UA, hisLG-DS, and hisLG-DA, respectively. Then, using the upstream and downstream homologous arms as templates, overlapping fragments were amplified by overlapping PCR using hisLG-US and hisLG-DA primers. Using hisLG-pGRB-S and hisLG-pGRB-A primers, gRNA fragments were annealed to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain hisLG-pGRB. E. coli W3110 electroporation competent cells were prepared, and the overlapping fragments and hisLG-pGRB were electroporated into competent cells together. Positive transformants were screened to obtain strain YTH1.

[0062] (2) Using the trc promoter at the rph pseudogene site to control the origin of Corynebacterium glutamicumoverexpression of hisG* from Corynebacterium glutamicum ATCC 13032: the same operation method as in step (2) was used, and the primers used were yeeP-U-S, yeeP-U-A, hisG-1-S, hisG-1-A, hisG-2-S, hisG-2-A, yeeP-D-S, yeeP-D-A, yeeP-pGRB-S, and yeeP-pGRB-A; the competent cells were YTH2 to obtain strain YTH3. hisG * the target gene fragment, and finally, the upstream homologous arm, the downstream homologous arm, and the target gene fragment were used as templates, and rph-U-S and rph-D-A were used as primers to obtain an overlapping fragment by overlapping PCR. The gene integration fragment Ptrc-hisG* (rph) was obtained by overlapping PCR, and the gene integration fragment was composed of the rph upstream homologous arm, the Ptrc-hisG* target gene, and the rph downstream homologous arm; the gRNA fragment containing the target sequence was obtained by a PCR annealing program using rph-pGRB-S and rph-pGRB-A as primers, and was ligated with the pGRB vector to obtain rph-pGRB; YTH5 competent cells were prepared, the overlapping fragment and rph-pGRB were electroporated into the competent cells, and positive transformants were screened to obtain strain YTH2.

[0063] (3) Overexpression of hisG* from Corynebacterium glutamicum ATCC 13032 at the yeeP pseudogene site using a trc promoter: the same operation method as in step (2) was used, and the primers used were yeeP-U-S, yeeP-U-A, hisG-1-S, hisG-1-A, hisG-2-S, hisG-2-A, yeeP-D-S, yeeP-D-A, yeeP-pGRB-S, and yeeP-pGRB-A; the competent cells were YTH2 to obtain strain YTH3. Corynebacterium glutamicum (4) Overexpression of hisG* from Corynebacterium glutamicum ATCC 13032 at the yghX pseudogene site using a trc promoter: the same operation method as in step (2) was used, and the primers used were yghX-U-S, yghX-U-A, hisG-1-S, hisG-1-A, hisG-2-S, hisG-2-A, yghX-D-S, yghX-D-A, yghX-pGRB-S, and yghX-pGRB-A; the competent cells were YTH3 to obtain strain YTH4.

[0064] Corynebacterium glutamicum (4) Overexpression of hisG* from Corynebacterium glutamicum ATCC 13032 at the yghX pseudogene site using a trc promoter: the same operation method as in step (2) was used, and the primers used were yghX-U-S, yghX-U-A, hisG-1-S, hisG-1-A, hisG-2-S, hisG-2-A, yghX-D-S, yghX-D-A, yghX-pGRB-S, and yghX-pGRB-A; the competent cells were YTH3 to obtain strain YTH4. ​

[0065] (5) Integration of the histidine operon hisDCBHAFI genes:

[0066] Since the full-length of the histidine operon hisDCBHAFI genes in the W3110 strain genome is 6405 bp, they are integrated into the genome of the W3110 strain by the method of segmented integration (for details, refer to CN114774341A specification

[0096] paragraph) in sequence E.coli The four segments are hisD ilvG hisCB 、 hisHA 、 hisFI. 、

[0067] ① Integration of the gene hisD :

[0068] With the W3110 genome as the template, the upstream and downstream homologous arms were obtained by pcr amplification with ilvG-U-S, ilvG-U-A and ilvG-D-S-1#, ilvG-D-A, respectively; with the W3110 genome as the template, the target gene fragment was obtained by pcr amplification with hisD-S, hisD-A-1# as primers; then, with the upstream and downstream homologous arms and the target gene fragment as the template, the overlapping fragment was obtained by overlapping pcr amplification with ilvG-U-S, ilvG-D-A as primers; since the Trc promoter has been connected to the 5' end of the primer during primer design, the overlapping fragment has the Trc promoter, that is, the upstream homologous arm-Trc promoter-target gene-downstream homologous arm overlapping fragment; with ilvG-pGRB-S, ilvG-pGRB-A as primers, the gRNA fragment was annealed and connected with the pGRB vector to obtain ilvG-pGRB; prepare YTH4 electrotransformation competent cells, and electrotransform the target fragment and ilvG-pGRB into the competent cells, and screen to obtain positive transformants to obtain strain YTH5-1. E.coli E.coli ② Integration of the gene :

[0069] hisCB

[0070] ​​​Upstream and downstream homologous arms were obtained by PCR amplification using hisCB-S, hisCB-A-4# and ilvG-DS-4#, ilvG-DA, respectively. Then, using the upstream and downstream homologous arms as templates, overlapping fragments were obtained by overlapping PCR amplification using hisCB-S and ilvG-DA primers. The gRNA fragment was obtained by annealing using pGRB-1#-S and pGRB-1#-A primers and ligated into the pGRB vector to obtain pGRB-1#. YTH5-1 electroporation competent cells were prepared, and the target fragment and pGRB-1# were electroporated into competent cells together. Positive transformants were screened to obtain strain YTH5-2.

[0071] ③Genes hisHA Integration:

[0072] respectively hisHA -S、 hisHA -A-1# and ilvG-DS-1#, ilvG-DA were amplified by PCR to obtain upstream and downstream homologous arms. Then, using the upstream and downstream homologous arms as templates, hisHA Using primers -S and ilvG-DA, overlapping fragments were amplified by overlapping PCR; using primers pGRB-4#-S and pGRB-4#-A, gRNA fragments were annealed and ligated with the pGRB vector to obtain pGRB-1#; YTH5-2 electrotransformation competent cells were prepared, and the target fragment and pGRB-4# were electrotransformed together into competent cells, and positive transformants were screened to obtain strain YTH5-3.

[0073] ④Genes hisFI Integration:

[0074] respectively hisFI -S、 hisFI -A and ilvG-DS, ilvG-DA were amplified by PCR to obtain upstream and downstream homologous arms. Then, using the upstream and downstream homologous arms as templates, hisFI Using primers -S and ilvG-DA, overlapping fragments were amplified by overlapping PCR; using primers pGRB-1#-S and pGRB-1#-A, gRNA fragments were annealed and ligated with the pGRB vector to obtain pGRB-1#; YTH5-3 electrotransformation competent cells were prepared, and the target fragment and pGRB-4# were electrotransformed into competent cells together, and positive transformants were screened to obtain strain YTH5.

[0075] (6) Overexpression of the artificial operon rpiAB gene at the ycgH pseudogene site using the trc promoter: using ycgH-US, ycgH-UA, ycgH-DS, and ycgH-DA as primers, upstream and downstream homologous arms were obtained by PCR amplification. Using rpiA-S, rpiA-A, rpiB-S, and rpiB-A as primers, rpiA and rpiB fragments were obtained by PCR amplification. Then, using rpiA and rpiB fragments as templates, and using rpiA-S and rpiB-A as primers, the artificial operon was obtained by overlap PCR amplification. rpiAB The target gene fragment was amplified by overlapping PCR using the upstream and downstream homologous arms and the target gene fragment as templates and ycgH-US and ycgH-DA as primers. The overlapping fragment was then obtained by overlapping PCR to obtain the integrated fragment of the artificial operon Ptrc-rpiAB (ycgH) gene. The gRNA fragment was annealed using ycgH-pGRB-S and ycgH-pGRB-A primers and ligated into the pGRB vector to obtain ycgH-pGRB. YTH5 electroporation competent cells were prepared by electroporating the target fragment and ycgH-pGRB together into competent cells, and positive transformants were screened to obtain strain YTH6.

[0076] (7) Using the trc promoter at the yeeL pseudogene site to control the origin of Corynebacterium glutamicum zwf* overexpression: The same operation method as in step (2) was used, and the primers used were yeeL-US, yeeL-UA, zwf*-1-S, zwf*-1-A, zwf*-2-S, zwf*-2-A, yeeL-DS, yeeL-DA, yeeL-pGRB-S and yeeL-pGRB-A. The competent cells were YTH6 obtained from strain YTH7.

[0077] (8) Using the trc promoter at the ygaY pseudogene site to control the origin of Corynebacterium glutamicum overexpression of gnd*: The same operation method as in step (2) was used, and the primers used were ygaY-US, ygaY-UA, gnd*-1-S, gnd*-1-A, gnd*-2-S, gnd*-2-A, ygaY-DS, ygaY-DA, ygaY-pGRB-S and ygaY-pGRB-A. The competent cells were YTH7 and the strain YTH8 was obtained.

[0078] (9) Overexpression of prsA* at yghE pseudogene locus using trc promoter: the same operation method as in step (2) was used, and the primers used were yghE-U-S, yghE-U-A, prsA*-l-S, prsA*-l-A, prsA*-2-S, prsA*-2-A, yghE-D-S, yghE-D-A, yghE-pGRB-S, and yghE-pGRB-A. The YTH9 strain was obtained using YTH8 as the competent cell.

[0079] (10) Knockout of ushA gene: the same operation method as in step (1) was used, except that the primers used were ushA-U-S, ushA-U-A, ushA-D-S, ushA-D-A, ushA-pGRB-S, and ushA-pGRB-A. The YTH10 strain was obtained using YTH9 as the competent cell.

[0080] (11) Knockout of surE gene: the same operation method as in step (1) was used, except that the primers used were surE-U-S, surE-U-A, surE-D-S, surE-D-A, surE-pGRB-S, and surE-pGRB-A. The YTH11 strain was obtained using YTH10 as the competent cell.

[0081] (12) Knockout of nagD gene: the same operation method as in step (1) was used, except that the primers used were nagD-U-S, nagD-U-A, nagD-D-S, nagD-D-A, nagD-pGRB-S, and nagD-pGRB-A. The YTH12 strain was obtained using YTH11 as the competent cell.

[0082] (13) Knockout of nrdD gene: the same operation method as in step (1) was used, except that the primers used were nrdD-U-S, nrdD-U-A, nrdD-D-S, nrdD-D-A, nrdD-pGRB-S, and nrdD-pGRB-A. The YTH13 strain was obtained using YTH12 as the competent cell.

[0083] Example 2

[0084] This example aims to illustrate the shake flask fermentation application of the strain YTH13 obtained in Example 1, and the specific steps are as follows:

[0085] ① Inoculation on slant: the strain YTH13 preserved in a -80°C refrigerator was inoculated on a slant culture medium, and incubated at 37°C for 12 h. The slant culture medium used was a general LB solid culture medium.

[0086] ② Shake-flask seed culture: Solid slant culture was inoculated into shake-flask culture medium for fermentation. The culture temperature was 37℃, the culture time was 12 hours, the shaker speed was 220 r / min, and the pH was 7.0. The seed culture medium used was:

[0087] Glucose 30 g / L, yeast powder 6 g / L, peptone 2 g / L, MgSO4·7H2O 0.8 g / L, KH2PO4·3H2O 2 g / L, ammonium sulfate 1 g / L, FeSO4·7H2O 10 mg / L, threonine 1 g / L, methionine 0.5 g / L, citric acid 2 g / L, the remainder being water;

[0088] ③ Shake-flask fermentation culture: The inoculum size was 20%-25%, the culture temperature was 37℃, the pH was 7.0, the culture time was 24h, and the shaker speed was 220r / min. The fermentation medium used was: MgSO4·7H2O 1.5g / L, yeast powder 5g / L, peptone 2g / L, ammonium sulfate 3g / L, methionine 0.2g / L, K2HPO4·3H2O 4g / L, citric acid 2.5g / L, threonine 1g / L, lysine 1g / L, isoleucine 0.5g / L, FeSO4·7H2O 20mg / L, MnSO4 20mg / L, MgSO4·7H2O 1.5g / L, VB1, VB3, and VB5 2mg / L each, and the remainder was water.

[0089] wild type E.coli W3110 served as the control group. After 24 hours of fermentation, the wild-type was verified. E.coli W3110 failed to accumulate L-histidine, while the engineered strain YTH13 described in Example 1 accumulated 10.6 g / L of L-histidine, demonstrating the effectiveness of the strain.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. An L-histidine-producing strain, characterized in that: by E. coli W3110, as a chassis strain, lacks the hisLG, ushA, surE, nagD, and nrdD genes, and heterologously expresses genes derived from... Corynebacterium glutamicum The hisG*, zwf*, and gnd* genes of ATCC 13032 were overexpressed, along with hisDCBHAFI, prsA*, and the artificial operon rpiAB gene. The nucleotide sequences of the hisG* gene, zwf* gene, gnd* gene, prsA* gene, hisDCBHAFI gene, and rpiAB gene were overexpressed. The nucleotide sequences of the hisG* gene, zwf* gene, gnd* gene, prsA* gene, and rpiAB gene were overexpressed.

2. The L-histidine-producing strain according to claim 1, characterized in that: by E. coli W3110, as a chassis strain, was obtained by modifying it using the following method: the hisLG, ushA, surE, nagD, and nrdD genes were knocked out from the strain's genome, and the trc promoter was used to enhance the expression of the strain derived from... Corynebacterium glutamicum The hisG* gene is transcribed and integrated into the rph gene locus in the genome, with three copies inserted into yeeP and yghX. The TRC promoter was used to enhance the source Corynebacterium glutamicum The zwf* gene was transcribed and integrated into the yeeL gene locus in the genome; the trc promoter was used to enhance the expression of the zwf* gene. Corynebacterium glutamicum The gnd* gene was transcribed and integrated into the ygaY pseudogene locus in the genome; the prsA* gene was transcribed using the trc promoter and integrated into the yghE pseudogene locus in the genome; the hisDCBHAFI gene was transcribed using the trc promoter and integrated into the ilvG pseudogene locus in the genome. The transcription of the artificial operon rpiAB gene was enhanced using the trc promoter and integrated into the genomic ycgH pseudogene locus.

3. The L-histidine-producing strain according to claim 1 or 2, characterized in that: The nucleotide sequence of the hisLG gene is shown in SEQ ID NO.1, the nucleotide sequence of the ushA gene is shown in SEQ ID NO.2, the nucleotide sequence of the surE gene is shown in SEQ ID NO.3, the nucleotide sequence of the ngD gene is shown in SEQ ID NO.4, and the nucleotide sequence of the nrdD gene is shown in SEQ ID NO.

5.

4. The L-histidine-producing strain according to claim 2, characterized in that: The nucleotide sequence of the trc promoter is shown in the sequence listing SEQ ID NO.

13.

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

6. The method for constructing the L-histidine-producing strain according to any one of claims 1-5, characterized in that: In the originating strain E. coli The following are the specific steps for targeted modification based on the W3110: (1) Knock out hisLG; introduce from Corynebacterium glutamicum the hisG* gene; (2) hisDCBHAFI was overexpressed using the trc promoter; (3) The prsA* and rpiAB genes were overexpressed using the trc promoter; genes derived from... Corynebacterium glutamicum The zwf* and gnd* genes; (4) Knock out the ushA, surE, nagD, and nrdD genes.

7. The use of the L-histidine producing strain according to any one of claims 1-5 in the fermentation production of L-histidine.

8. The application according to claim 7, characterized in that: The specific steps are as follows: ① Slant culture: The L-histidine producing strain was inoculated onto a slant culture medium for culture; ② Shake flask seed culture: Take solid slant inoculum and inoculate it into shake flask culture medium for fermentation; ③ Shake-flask fermentation culture: The inoculum size is 20%-25%.

9. The application according to claim 8, characterized in that: The slant culture medium used in step ① is general-purpose LB solid medium; the seed culture medium used in step ② is: glucose 30 g / L, yeast extract 6 g / L, peptone 2 g / L, MgSO4·7H2O 0.8 g / L, KH2PO4·3H2O 2 g / L, ammonium sulfate 1 g / L, FeSO4·7H2O 10 mg / L, threonine 1 g / L, methionine 0.5 g / L, citric acid 2 g / L, with the remainder being water; the fermentation culture medium used in step ③ is: MgSO4·7H2O 1.5 g / L, yeast extract 5 g / L, peptone 2 g / L, ammonium sulfate 3 g / L, methionine 0.2 g / L, K2HPO4·3H2O 4 g / L, citric acid 2.5 g / L, threonine 1 g / L, lysine 1 g / L, isoleucine 0.5 g / L, FeSO4·7H2O 20 mg / L, MnSO4 20 mg / L, MgSO4·7H2O 1.5 g / L, VB1, VB3, and VB5 2 mg / L each, with the remainder being water.

Images