L-histidine production strain as well as construction method and application thereof

Abstract

The invention provides a strain for producing L-histidine as well as a construction method and application of the strain. According to the strain, E.coli W3110 is taken as a chassis strain, hisLG, ushA, surE, nagD and nrdD genes are deleted, hisG *, zwf * and gnd * genes derived from Cornebacterium glutamicum ATCC 13032 are subjected to heterologous expression by using a trc promoter, and meanwhile, hisDCBHAFI, prsA * and artificial operon rfiAB genes are subjected to overexpression by using the trc promoter; the strain can effectively improve the synthesis efficiency of L-histidine by relieving feedback inhibition of key enzymes, knocking out branch pathway genes synthesized by ATP, enhancing expression of pathway key enzymes and increasing the content of PRPP, is stable in heredity, does not need to add resistant substances, and has good synthesis capacity of L-histidine and high acid production efficiency.

Description

Technical Field

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

[0002] Histidine is one of the 20 standard protein-forming amino acids and is involved in protein synthesis. L-histidine is widely used in various fields, including biomedicine, and has diverse physiological functions, making it a popular choice for food, medicine, and other applications.

[0003] Currently, the main methods for producing L-histidine can be divided into protein hydrolysis, chemical synthesis, and microbial fermentation. Although protein hydrolysis is a mature process, its application and development are significantly limited by severe equipment corrosion, high hydrolysis losses, low yields, and significant environmental pollution. Chemical synthesis involves complex production steps and long production cycles, and the resulting product is often a DL racemate. Microbial fermentation utilizes microorganisms to convert relatively inexpensive industrial raw materials into the target product. Compared to protein hydrolysis and chemical synthesis, microbial fermentation offers milder conditions, less environmental pollution, easier process control, and readily available fermentation raw materials, making it the preferred method for future industrial production of histidine.

[0004] Despite the rapid development of microbial fermentation in recent years, histidine production still faces challenges such as low yield, low sugar-to-acid conversion, high production costs, and long fermentation cycles. Therefore, constructing an L-histidine-producing strain with high acid production efficiency and good genetic stability is a pressing technical challenge. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an L-histidine producing strain.

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

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

[0008] In order to solve the above technical problems, the technical solution of the present invention is: An artificial operon is named artificial operon rpiAB, and its nucleotide sequence is shown in the sequence listing as SEQ ID NO.16.

[0009] Preferably, the artificial operon includes the rpiA gene and the rpiB gene, specifically: (1) The operon gene sequence is trc promoter, rpiA, RBS1, rpiB, rrnB T1 terminator; (2) The trc promoter is directly linked to the rpiA gene from head to tail; (3) rpiA and rpiB are linked by the RBS1 gene; (4) The rpiB and rrnB T1 terminators are directly connected end to end.

[0010] Preferably, in the above-mentioned artificial operon, the nucleotide sequence of the rpiA gene is shown in the sequence listing SEQ ID NO.11, the nucleotide sequence of the rpiB gene is shown in the sequence listing SEQ ID NO.12, the nucleotide sequence of the RBS1 gene is shown in the sequence listing SEQ ID NO.14, and the nucleotide sequence of the rrnB T1 terminator is shown in the sequence listing SEQ ID NO.15.

[0011] The above-mentioned artificial operon construction method is achieved by optimizing the gene connection mode and the gene position in the operon, wherein the transcription of the operon is controlled by the same trc promoter and the transcription of the operon is controlled by the same rrnB T1 terminator.

[0012] An L-histidine producing strain, named strain YTH13, has all the characteristics of the above artificial operon in its genome. E.coli W3110 was used as the chassis strain, and the hisLG, ushA, surE, nagD, and nrdD genes were deleted. The trc promoter was used to heterologously express the Corynebacterium glutamicum The hisG*, zwf*, and gnd* genes of ATCC 13032 were overexpressed, and the hisDCBHAFI, prsA*, and artificial operon rpiAB genes were overexpressed using the trc promoter.

[0013] Preferably, the above-mentioned L-histidine producing strain is E.coli W3110 was used as the chassis strain and was transformed by the following method: the hisLG, ushA, surE, nagD, and nrdD genes on the strain genome were knocked out, and the trc promoter was used to enhance the expression of Corynebacterium glutamicum The hisG* gene is transcribed and integrated into the genomic rph gene site and three copies are added to yeeP and yghX; the trc promoter is used to enhance the expression of Corynebacterium glutamicum The zwf* gene is transcribed and integrated into the yeeL gene site of the genome; the trc promoter is used to enhance the expression of Corynebacterium glutamicumThe gnd* gene transcription was enhanced and integrated into the genomic ygaY pseudogene site; the prsA* gene transcription was enhanced using the trc promoter and integrated into the genomic yghE pseudogene site; the hisDCBHAFI gene transcription intensity was enhanced using the trc promoter and integrated into the genomic ilvG pseudogene site; the artificial operon rpiAB gene transcription was enhanced using the trc promoter and integrated into the genomic ycgH pseudogene site.

[0014] Preferably, in the above-mentioned L-histidine-producing strain, the nucleotide sequence of the hisLG gene is shown in the sequence listing SEQ ID NO.1, the nucleotide sequence of the ushA gene is shown in the sequence listing SEQ ID NO.2, the nucleotide sequence of the surE gene is shown in the sequence listing SEQ ID NO.3, the nucleotide sequence of the nagD gene is shown in the sequence listing SEQ ID NO.4, and the nucleotide sequence of the nrdD gene is shown in the sequence listing SEQ ID NO.5.

[0015] Preferably, in the above-mentioned L-histidine-producing strain, the nucleotide sequence of the hisG* gene is shown in the sequence listing SEQ ID NO.6, the nucleotide sequence of the zwf* gene is shown in the sequence listing SEQ ID NO.7, the nucleotide sequence of the gnd* gene is shown in the sequence listing SEQ ID NO.8, the nucleotide sequence of the prsA* gene is shown in the sequence listing SEQ ID NO.9, the nucleotide sequence of the hisDCBHAFI gene is shown in the sequence listing SEQ ID NO.10, and the nucleotide sequence of the trc promoter is shown in the sequence listing SEQ ID NO.13.

[0016] Preferably, the above-mentioned L-histidine producing strain, E.coli W3110 is E.coli W3110 ATCC 27325.

[0017] The above-mentioned method for constructing the L-histidine production strain is to start with the strain E.coli Directional transformation is carried out based on W3110. The specific steps are as follows: (1) Chassis bacteria transformation: release L-histidine feedback regulation: knock out hisLG; introduce Corynebacterium glutamicum hisG* gene; (2) Enhanced expression of key enzymes in the pathway: Using the trc promoter, hissDCBHAFI was overexpressed, resulting in efficient expression of key enzymes in the L-histidine metabolic pathway, thereby greatly increasing the production of L-histidine; (3) Enhance the synthesis of the precursor PRPP: Use the trc promoter to overexpress the prsA* and artificial operon rpiAB genes; introduce Corynebacterium glutamicumThe zwf* and gnd* genes increase the supply of precursor substances, which is beneficial to the efficient production of the final product; (4) Enhance ATP synthesis: Knock out the ushA, surE, nagD, and nrdD genes to enhance energy supply and facilitate efficient production of the final product.

[0018] Application of the L-histidine producing strain in the fermentation production of L-histidine.

[0019] The L-histidine-producing strain is used to ferment L-histidine in a culture medium under suitable fermentation conditions. The culture medium includes, but is not limited to, a carbon source, a nitrogen source, inorganic salts, vitamins, etc.; and the fermentation conditions include, but are not limited to, fermentation temperature, fermentation pH, fermentation dissolved oxygen conditions, fermentation pressure, and fermentation time. The culture medium can be obtained by conventional methods and used for L-histidine production. The fermentation conditions can be adjusted to suit the production characteristics of the strain.

[0020] Preferably, the application of the above-mentioned L-histidine production strain comprises the following specific steps: ① Slant culture: Take the L-histidine producing strain and inoculate it on the slant culture medium for culture; ② Shake flask seed culture: Take the solid slant bacteria and inoculate them into the shake flask culture medium for fermentation; ③ Shake flask fermentation culture: the fermentation inoculation amount is 20%-25%.

[0021] Preferably, the application of the above-mentioned L-histidine production strain comprises the following specific steps: ① Slant culture: inoculate the L-histidine-producing strain onto a slant culture medium and culture at 37°C for 12 hours; ② Shake flask seed culture: Take the solid slant bacteria and inoculate them into the shake flask medium for fermentation. The culture temperature is 37℃, the culture time is 12h, the shaker speed is 220r / min, and the pH is 7.0. ③ Shake flask fermentation culture: the fermentation inoculation amount is 20%-25%, the culture temperature is 37℃, pH 7.0, the culture time is 24h, and the shaker speed is 220r / min.

[0022] Preferably, in the application of the above-mentioned L-histidine-producing strain, the slant culture medium used in step ① is a universal LB solid culture medium.

[0023] Preferably, in the application of the above-mentioned L-histidine-producing strain, the seed culture medium used in step ② is: glucose 30 g / L, yeast powder 6 / 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, and the rest is water.

[0024] Preferably, in the application of the above-mentioned L-histidine-producing strain, the fermentation medium used in step ③ is: MgSO4·7H2O 1.5g / L, yeast powder 5g / L, peptone 2g / L, ammonium sulfate 3g / L, methionine 0.2g / L, K2HPO4·3H2O4g / L, citric acid 2.5g / L, threonine 1g / L, lysine 1g / L, isoleucine 0.5g / L, FeSO4·7H2O 20mg / L, MnSO420mg / L, MgSO4·7H2O 1.5g / L, VB1, VB3, VB5 each 2mg / L, and the rest is water.

[0025] The above culture media can be prepared using standard methods.

[0026] Beneficial effects: The L-histidine production strain described above does not contain plasmids, is genetically stable, does not require the addition of resistance substances, has good L-histidine synthesis capacity, low production cost, stable performance, high acid production efficiency, and good economic benefits. By relieving feedback inhibition of key enzymes, knocking out genes in the branch pathway of ATP synthesis, enhancing the expression of key enzymes in the pathway, and increasing PRPP content, the strain can effectively improve the efficiency of L-histidine synthesis and the level of L-histidine production, achieving efficient production of L-histidine and having excellent industrial application prospects. Specifically: (1) By knocking out hisL, the inhibition of histidine structure gene transcription was lifted; by knocking out hisG, the Corynebacterium glutamicum The hisG* gene was removed from the trc promoter to overexpress hisDCBHAFI, which resulted in efficient expression of key enzymes in the L-histidine metabolic pathway, thereby greatly increasing the production of L-histidine. (2) By knocking out the ushA, surE, nagD, and nrdD genes, the metabolism of the ATP synthesis branch pathway is reduced, the energy supply is enhanced, and the efficient production of the final product is facilitated; (3) Overexpression of prsA* and rpiAB genes by using the trc promoter; introduction of Corynebacterium glutamicum The zwf* and gnd* genes increase the synthesis of the precursor PRPP, thereby greatly improving the production of L-histidine. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Diagram of the genetic modification process of the de novo synthesis pathway of L-histidine-producing strain; wherein, GUO: 5-(5-phospho-D-ribosylaminoformyl)-1-(5-phosphoribosyl)-imidazole-4-carboxamide; 2ER: 5-[(5-phospho-1-deoxyribose-1-amino)methylacetamide]-1-(5-phosphoribosyl)imidazole-4-carboxamide. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0029] The percentage sign "%" involved in the examples, unless otherwise specified, refers to mass percentage. The percentage of a solution refers to the number of grams of solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0030] The starting strain used in the embodiment is the wild type E.coli W3110 ATCC 27325 (commercially available), corresponding promoters and genes are listed in the sequence listing. Primers used in the construction of the strains involved are listed in Table 1.

[0031] Table 1 Primers involved in strain construction Primer Name Sequence Number Primer Sequence (5'-3') hisLG-pGRB-S SEQ ID NO.17 AGTCCTAGGTATAATACTAGTCGGCGGCTGTCGTCTTTCGCGTTTTAGAGCTAGAA hisLG-pGRB-A SEQ ID NO18 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 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGCTCATCTTTGCGGGCTT -D-S SEQ ID NO.43 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATATCCGCAAGCGACAGGC -D-A SEQ ID NO.44 CGTTGATTCGGGTGTCCAG -pGRB-S SEQ ID NO.45 AGTCCTAGGTATAATACTAGTTATCGGCACTGACGCATTTCGTTTTAGAGCTAGAA -pGRB-A SEQ ID NO.46 AGTCCTAGGTATAATACTAGTGGAAGAGTTGCCGCGCATCAGTTTTAGAGCTAGAA -U-S SEQ ID NO.47 ACCGAGGAGCAGACAATGAATAA -U-A SEQ ID NO.48 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 GCCCCAAGGGGTTATGCTAGCCTACAAATTGAGTTATGTTCATTTAAATATGTTGTTCAGGCAGTTCAATACCTTCGTCCGC 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 GCCCCAAGGGGTTATGCTAGCCTACAAATTGAGTTATGTTCATTTAAATATGTTGTTCAGGGAGGACTGAAATGCCACCTG hisFI-S SEQ ID NO.59 GAGAACTCGGGCGTTTCACT hisFI-A SEQ ID NO.60 caccgacaaacaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttGTCACTGATGCCGTTTACGCAG SEQ ID NO.61 AGTCCTAGGTATACTAGTATGAACATAACTCAATTTGTGTTTTAGAGCTAGAA SEQ ID NO.62 TTCTAGCTCTAAAACAAAATTTGAGTTATGTTCATACTAGTATTATACCTAGGACT SEQ ID NO.63 AGTCCTAGGTATACTAGTTGCGCTGGTTGATTTCTTCTGTTTTAGAGCTAGAA SEQ ID NO.64 TTCTAGCTCTAAAACAGAAGAAATCAACCAGCGCAACTATTTATACCTAGGACT -pGRB-S SEQ ID NO.65 AGTCCTAGGTATACTAGTTATGCGTCTGAACGACCGTGGTTTTAGAGCTAGAA -pGRB-A SEQ ID NO.66 TTCTAGCTCTAAAACCACGGTCGTTCAGACGCATAACTAGTATTATACCTAGGACT -US SEQ ID NO.67 TAAACTCGTCAGCGGCCACAAC -UA SEQ ID NO.68 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGTAGGCGTTTCTGTTGATTCTG -D-S SEQ ID NO.69 GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCGTGTCGGATTATCGTTCG -D-A SEQ ID NO.70 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 -1-S SEQ ID NO.81 TGTGGAATTGTGAGCGGATAACAATTTCACAGGAAACAGACCgtgagcacaaacgacccc -1-A SEQ ID NO.82 acccaagccaatatcttcagtcatg -2-S SEQ ID NO.83 catgactgaagatattggcttgggt -2-A SEQ ID NO.84 caacagataaaacgaaaggcccagtctttcgactgagccttcgttttatttGttatggcctgcgccaggt -pGRB-S SEQ ID NO.85 AGTCCTAGGTATAATACTAGTCACTGATGGCGCTGGCATTAGTTTTAGAGCTAGAA -pGRB-A SEQ ID NO.86 TTCTAGCTCTAAAACTAATGCCAGCGCCATCAGTGACTAGTATTATACCTAGGACT -US SEQ ID NO.87 CCTACAAACCACATCGCACATT -UA SEQ ID NO.88 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAACACCGAAGCAACCCAAAG -DS SEQ ID NO.89 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTGCTTGCCGCTCCACC -YES SEQ ID NO.90 GGAGTAGGGCTTTCCATAGAGTGT -1-S SEQ ID NO.91 TGTGGAATTGTGAGCGGATAACAATTTCACAGGAAACAGACCatgccgtcaagtacgatcaataacatg -1-A SEQ ID NO.92 ttctcgtcgaagccagcctt -2-S SEQ ID NO.93 aaggctggcttcgacgagaa -2-A SEQ ID NO.94 cagataaaacgaaggcccagtctttcgactgagccttcgttttatttGttaagcttcaacctcggagcg 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 like Figure 1 As shown, the strain was transformed according to the metabolic pathway of the entire strain transformation process to obtain the L-histidine-producing strain YTH13. The transformation process mainly includes the following four modules: (1) Chassis bacteria transformation: release L-histidine feedback regulation: knock out hisLG; introduce Corynebacterium glutamicum hisG* gene; (2) Enhanced expression of key enzymes in the pathway: Using the trc promoter, hissDCBHAFI was overexpressed, resulting in efficient expression of key enzymes in the L-histidine metabolic pathway, thereby greatly increasing the production of L-histidine; (3) Enhance the synthesis of the precursor PRPP: Use the trc promoter to overexpress the prsA* and artificial operon rpiAB genes; introduce Corynebacterium glutamicum The zwf* and gnd* genes increase the supply of precursor substances, which is beneficial to the efficient production of the final product; (4) Enhance ATP synthesis: Knock out the ushA, surE, nagD, and nrdD genes to enhance energy supply and facilitate efficient production of the final product.

[0032] The gene editing method used in the above gene manipulation is based on the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli Using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21. Unless otherwise noted, all technical terms used in this article are explained in that article. "Knockout" as used in this article refers to the inactivation of a target gene, and "introduction" refers to the insertion of a foreign gene into the genome of an engineered bacterium after ligating it to a promoter and terminator.

[0033] Example 1 This example is intended to illustrate the specific construction steps of strain YTH13. In particular, if there is a method for the same type of gene manipulation in the examples, only one of them will be provided and annotated without further elaboration.

[0034] The construction process of the L-histidine production strain comprises the following specific steps: (1) Knockout of hisLG gene: E.coli The W3110 genome was used as a template, and the upstream and downstream homology arms were amplified by PCR using hisLG-US, hisLG-UA and hisLG-DS, hisLG-DA, respectively. Then, the upstream and downstream homology arms were used as templates and hisLG-US and hisLG-DA were used as primers to amplify overlapping fragments by overlapping PCR. Using hisLG-pGRB-S and hisLG-pGRB-A as primers, the gRNA fragment was annealed to obtain the gRNA fragment, which was ligated with the pGRB vector to obtain hisLG-pGRB. E. coli W3110 electroporation competent cells were prepared, the overlapping fragments and hisLG-pGRB were electroporated into the competent cells together, and the positive transformants were screened to obtain the strain YTH1.

[0035] (2) Using the trc promoter at the rph pseudogene locus to control the expression of Corynebacterium glutamicumOverexpression of hisG*: Using the Escherichia coli W3110 genome as a template, rph-US, rph-UA, rph-DS, and rph-DA as primers, the upstream homology arm and downstream homology arm were amplified by PCR, and then using Corynebacterium glutamicum ATCC 13032 as a template, hisG-1-S, hisG-1-A, hisG-2-S, and hisG-2-A as primers, the hisG-1 and hisG-2 fragments were amplified by PCR, and then the hisG-1 and hisG-2 fragments were used as templates, hisG-1-S and hisG-2-A as primers, and the hisG-2 fragments were amplified by overlapping PCR. this * The target gene fragment was amplified by overlapping PCR using the upstream homology arm, downstream homology arm, and target gene fragment as templates and rph-US and rph-DA as primers to obtain an overlapping fragment. The Ptrc-hisG* (rph) gene integration fragment was obtained by overlapping PCR. The gene integration fragment consisted of the rph upstream homology arm, the Ptrc-hisG* target gene, and the rph downstream homology arm. Using rph-pGRB-S and rph-pGRB-A as primers, a gRNA fragment containing the target sequence was obtained by PCR annealing. This fragment was then ligated into the pGRB vector to obtain rph-pGRB. YTH5 electroporation competent cells were prepared, and the overlapping fragments and rph-pGRB were electroporated into the competent cells. Positive transformants were screened. The competent cells were designated YTH1 to obtain strain YTH2.

[0036] (3) Using the trc promoter at the yeeP pseudogene site to control the Corynebacterium glutamicum Overexpression of hisG*: The operation method is the same as in step (2), and the primers used are yeeP-US, yeeP-UA, hisG-1-S, hisG-1-A, hisG-2-S, hisG-2-A, yeeP-DS, yeeP-DA, yeeP-pGRB-S and yeeP-pGRB-A. The competent cell is YTH2 to obtain the strain YTH3.

[0037] (4) Using the trc promoter at the yghX pseudogene locus to control the Corynebacterium glutamicum Overexpression of hisG*: The operation method is the same as in (2), and the primers used are yghX-US, yghX-UA, hisG-1-S, hisG-1-A, hisG-2-S, hisG-2-A, yghX-DS, yghX-DA, yghX-pGRB-S and yghX-pGRB-A. The competent cell is YTH3 to obtain strain YTH4.

[0038] (5) Integration of the histidine operon hisDCBHAFI gene: Because in E.coli The histidine operon hisDCBHAFI gene in the W3110 strain genome is 6405 bp long and was sequentially integrated into the genome by a segmented integration method (for details, refer to paragraph

[0096] of the CN114774341A specification). ilvG site, these four segments are hisD 、 hisCB 、 hisHA 、 hisFI.

[0039] ①Gene this Integration of: by E.coli The W3110 genome was used as a template, and the upstream and downstream homology arms were obtained by PCR amplification using ilvG-US, ilvG-UA, ilvG-DS-1#, and ilvG-DA. E.coli W3110 was used as a template, hisD-S and hisD-A-1# were used as primers, and the target gene fragment was amplified by PCR; then, the upstream and downstream homology arms and the target gene fragment were used as templates, and ilvG-US and ilvG-DA were used as primers to obtain overlapping fragments by overlapping PCR. Since the Trc promoter was connected to the 5' end of the primer during primer design, the overlapping fragment contained the Trc promoter, that is, the upstream homology arm-Trc promoter-target gene-downstream homology arm overlapping fragment; using ilvG-pGRB-S and ilvG-pGRB-A as primers, the gRNA fragment was annealed and connected to the pGRB vector to obtain ilvG-pGRB; YTH4 electroporation competent cells were prepared, the target fragment and ilvG-pGRB were electroporated into the competent cells together, and positive transformants were screened to obtain strain YTH5-1.

[0040] ②Gene thisCB Integration of: The upstream and downstream homology arms were amplified by PCR using hisCB-S, hisCB-A-4# and ilvG-DS-4#, ilvG-DA, respectively. Then, the upstream and downstream homology arms were used as templates and hisCB-S and ilvG-DA were used as primers to obtain overlapping fragments by overlapping PCR amplification. Using pGRB-1#-S and pGRB-1#-A as primers, the gRNA fragment was annealed to obtain the gRNA fragment, which was ligated with the pGRB vector to obtain pGRB-1#. YTH5-1 electroporation competent cells were prepared, the target fragment and pGRB-1# were electroporated into the competent cells together, and the positive transformants were screened to obtain the strain YTH5-2.

[0041] ③Gene this Integration of: Respectively this -S, this -A-1# and ilvG-DS-1#, ilvG-DA were amplified by PCR to obtain the upstream and downstream homology arms. Then, the upstream and downstream homology arms were used as templates to this -S and ilvG-DA were used as primers to obtain overlapping fragments by overlapping PCR amplification; using pGRB-4#-S and pGRB-4#-A as primers, the gRNA fragment was annealed and connected with the pGRB vector to obtain pGRB-1#; YTH5-2 electroporation competent cells were prepared, the target fragment and pGRB-4# were electroporated into the competent cells together, and positive transformants were screened to obtain strain YTH5-3.

[0042] ④Gene this Integration of: Respectively this -S, this -A and ilvG-DS, ilvG-DA were amplified by PCR to obtain the upstream and downstream homology arms. Then, the upstream and downstream homology arms were used as templates to this -S and ilvG-DA were used as primers to obtain overlapping fragments by overlapping PCR amplification; using pGRB-1#-S and pGRB-1#-A as primers, the gRNA fragment was annealed and connected with the pGRB vector to obtain pGRB-1#; YTH5-3 electroporation competent cells were prepared, the target fragment and pGRB-4# were electroporated into the competent cells together, and positive transformants were screened to obtain strain YTH5.

[0043] (6) Using the trc promoter to control the overexpression of the artificial operon rpiAB gene at the ycgH pseudogene site: using ycgH-US, ycgH-UA, ycgH-DS, and ycgH-DA as primers, the upstream homology arm and the downstream homology arm were obtained by PCR amplification, and using rpiA-S, rpiA-A, rpiB-S, and rpiB-A as primers, the rpiA and rpiB fragments were obtained by PCR amplification. Then, using the rpiA and rpiB fragments as templates and rpiA-S and rpiB-A as primers, the artificial operon was obtained by overlapping PCR amplification. rpiABThe target gene fragment was amplified by overlapping PCR using the upstream homology arm, downstream homology arm and target gene fragment as templates and ycgH-US and ycgH-DA as primers. The artificial operon Ptrc-rpiAB (ycgH) gene integration fragment was obtained by overlapping PCR. ycgH-pGRB-S and ycgH-pGRB-A were used as primers to anneal the gRNA fragment, which was ligated with the pGRB vector to obtain ycgH-pGRB. YTH5 electroporation competent cells were prepared, the target fragment and ycgH-pGRB were electroporated into the competent cells together, and positive transformants were screened to obtain strain YTH6.

[0044] (7) Using the trc promoter at the yeeL pseudogene locus to control the Corynebacterium glutamicum Overexpression of zwf*: The operation method is the same as in step (2), and the primers used are 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 cell is YTH6 to obtain the strain YTH7.

[0045] (8) Using the trc promoter at the ygaY pseudogene locus to control the 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 cell was YTH7 to obtain the strain YTH8.

[0046] (9) Controlling prsA* overexpression using the trc promoter at the yghE pseudogene locus: The same operation method as in step (2) was used, and the primers used were yghE-US, yghE-UA, prsA*-1-S, prsA*-1-A, prsA*-2-S, prsA*-2-A, yghE-DS, yghE-DA, yghE-pGRB-S, and yghE-pGRB-A. The competent cell was YTH8, and the strain YTH9 was obtained.

[0047] (10) Knockout of the ushA gene: The same operation method as in step (1) was used, except that the primers used were ushA-US, ushA-UA, ushA-DS, ushA-DA, ushA-pGRB-S, and ushA-pGRB-A. The competent cell was YTH9, and the strain YTH10 was obtained.

[0048] (11) Knockout of the surE gene: The same procedure as in step (1) was used, except that the primers used were surE-US, surE-UA, surE-DS, surE-DA, surE-pGRB-S, and surE-pGRB-A. The competent cell was YTH10, and the strain YTH11 was obtained.

[0049] (12) Knockout of the nagD gene: The same procedure as in step (1) was used, except that the primers used were nagD-US, nagD-UA, nagD-DS, nagD-DA, nagD-pGRB-S, and nagD-pGRB-A. The competent cell was YTH11, and the strain YTH12 was obtained.

[0050] (13) Knockout of the nrdD gene: The procedure is the same as in step (1), except that the primers used are nrdD-US, nrdD-UA, nrdD-DS, nrdD-DA, nrdD-pGRB-S, and nrdD-pGRB-A. The competent cell is YTH12, and the strain YTH13 is obtained.

[0051] Example 2 This example is intended to illustrate the application of shake flask fermentation of strain YTH13 obtained in Example 1. The specific steps are as follows: ① Slant culture: The strain YTH13 stored in a -80°C refrigerator was inoculated on a slant culture medium and cultured at 37°C for 12 h. The slant culture medium used was the universal LB solid medium.

[0052] ② Shake flask seed culture: Take the solid slant bacteria and inoculate them into the shake flask medium for fermentation. The culture temperature is 37℃, the culture time is 12h, the shaker speed is 220r / min, the pH is 7.0, and the seed culture medium used is: Glucose 30 g / L, yeast powder 6 / 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, and the rest is water; ③ Shake flask fermentation culture: the fermentation inoculum size was 20%-25%, the culture temperature was 37°C, the pH was 7.0, the culture time was 24 h, and the shaker speed was 220 r / min; the fermentation medium used was: MgSO4·7H2O 1.5 g / L, yeast powder 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, VB5 2 mg / L each, and the rest was water.

[0053] Wild type E.coli W3110 was the control group, and after 24h fermentation verification, the wild type E.coli W3110 failed to accumulate L-histidine, while the engineered bacterium YTH13 described in Example 1 accumulated 10.6 g / L of L-histidine, demonstrating the effectiveness of the strain.

[0054] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. Improvements and modifications such as strain transformation carried out by technicians in this technical field based on the method of the present invention or on the basis of the method are considered to be within the scope of protection of the present invention.

Claims

1. An artificial operon, characterized in that: The nucleotide sequence is shown in the sequence listing as SEQ ID NO.

16.

2. An L-histidine-producing strain, characterized in that: by E. coli W3110 was used as the chassis strain, which lacked the hisLG, ushA, surE, nagD, and nrdD genes and heterologously expressed the Corynebacterium glutamicum The hisG*, zwf*, and gnd* genes of ATCC 13032 simultaneously overexpress hisDCBHAFI, prsA*, and the artificial operon rpiAB genes according to claim 1.

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

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

5.

5. The L-histidine producing strain according to claim 2 or 3, characterized in that: The nucleotide sequence of the hisG* gene is shown in the sequence listing SEQ ID NO.6, the nucleotide sequence of the zwf* gene is shown in the sequence listing SEQ ID NO.7, the nucleotide sequence of the gnd* gene is shown in the sequence listing SEQ ID NO.8, the nucleotide sequence of the prsA* gene is shown in the sequence listing SEQ ID NO.9, the nucleotide sequence of the hisDCBHAFI gene is shown in the sequence listing SEQ ID NO.10, and the nucleotide sequence of the trc promoter is shown in the sequence listing SEQ ID NO.

13.

6. The L-histidine producing strain according to claim 2 or 3, characterized in that: described E. coli W3110 is E. coli W3110 ATCC 27325.

7. The method for constructing the L-histidine-producing strain according to any one of claims 2 to 6, characterized in that: In the starting strain E. coli Directional transformation is carried out based on W3110. The specific steps are as follows: (1) Knockout hisLG; Introduce Corynebacterium glutamicum hisG* gene; (2) overexpressed hisDCBHAFI using the trc promoter; (3) The prsA* and rpiAB genes were overexpressed using the trc promoter; Corynebacterium glutamicum zwf* and gnd* genes; (4) Knockout of ushA, surE, nagD, and nrdD genes.

8. Use of the L-histidine-producing strain according to any one of claims 2 to 6 in the fermentation production of L-histidine.

9. The use according to claim 8, characterized in that: The specific steps are as follows: ① Slant culture: Take the L-histidine producing strain and inoculate it on the slant culture medium for culture; ② Shake flask seed culture: Take the solid slant bacteria and inoculate them into the shake flask culture medium for fermentation; ③ Shake flask fermentation culture: the fermentation inoculation amount is 20%-25%.

10. The use according to claim 9, characterized in that: The slant culture medium used in step ① is a universal LB solid culture medium; the seed culture medium used in step ② is: glucose 30 g / L, yeast powder 6 / 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, and the rest is water; the fermentation medium used in step ③ is: MgSO4·7H2O 1.5 g / L, yeast powder 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 20mg / L, MnSO420mg / L, MgSO4·7H2O 1.5g / L, VB1, VB3, VB5 2mg / L each, and the rest is water.

Images