WO2024000364A1 - 一种谷氨酸棒杆菌高效突变体及重组菌构建方法与应用 - Google Patents

一种谷氨酸棒杆菌高效突变体及重组菌构建方法与应用 Download PDF

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WO2024000364A1
WO2024000364A1 PCT/CN2022/102703 CN2022102703W WO2024000364A1 WO 2024000364 A1 WO2024000364 A1 WO 2024000364A1 CN 2022102703 W CN2022102703 W CN 2022102703W WO 2024000364 A1 WO2024000364 A1 WO 2024000364A1
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mutant
seq
cgl1289m
mutation
ugi
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饶志明
李翔飞
乔郅钠
王晴
徐美娟
杨套伟
张显
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江南大学
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium

Definitions

  • the invention belongs to the technical fields of genetic engineering and mutagenesis, and in particular relates to a method and application for constructing a high-efficiency mutant and recombinant strain of Corynebacterium glutamicum.
  • the mutation methods for Corynebacterium glutamicum are still relatively traditional mutagenesis methods, such as ultraviolet mutagenesis, ARTP mutagenesis, chemical mutagenesis, etc., which are time-consuming and labor-intensive, and the mutation frequency is not high.
  • Ultraviolet mutagenesis can cause random mutations in the genome and is simple to operate. However, it has a certain sequence preference and the mutation efficiency is low. Moreover, the ultraviolet rays during the mutagenesis process can easily cause damage to the skin of the experimenter.
  • ARTP mutagenesis has a higher mutation rate and wider mutation spectrum, and is simple to operate.
  • ARTP mutagenesis requires a large amount of active particles (such as helium particles) and consumes energy.
  • Chemical mutagens such as ethyl methanesulfonate and 1-methyl-3-nitro-1-nitrosoguanidine can be used to construct mutant libraries and strain selection, but mutations caused by chemical mutagenesis
  • the spectrum is relatively narrow, and the chemical mutagenic reagents used are usually highly toxic substances and powerful human carcinogens.
  • Conventional laboratory evolutionary mutation methods have certain drawbacks. Therefore, the search for a random mutation system with milder mutation conditions, environmental protection, health and safety, and higher mutation efficiency and mutation spectrum has attracted widespread attention.
  • the present invention provides a method and application for constructing high-efficiency mutants and recombinant bacteria of Corynebacterium glutamicum.
  • the technical solution adopted by the present invention is:
  • the invention provides a high-efficiency mutant of Corynebacterium glutamicum, which includes any one of MP1 to MP5 and MP5T:
  • the MP1 includes DNA polymerase Cgl1289M, and the amino acid sequence of the DNA polymerase Cgl1289M is shown in SEQ ID NO: 13;
  • the MP2 includes the following elements: the Cgl1289M and the uracil DNA glycosylase inhibitor UGI; the downstream tandem UGI of the Cgl1289M, and the amino acid sequence of the UGI is as shown in SEQ ID NO: 25;
  • the MP3 includes the following elements: the Cgl1289M, UGI and cytosine deaminase pmCDA1; the Cgl1289M, UGI and pmCDA1 are connected in series in sequence; the amino acid sequence of the pmCDA1 is as shown in SEQ ID NO: 27;
  • the MP4 includes the following elements: the Cgl1289M, UGI and adenine deaminase TadA-ABE8e; the Cgl1289M, UGI and TadA-ABE8e are connected in series in sequence, and the amino acid sequence of the TadA-ABE8e is as shown in SEQ ID NO: 29 .
  • the MP5 includes the following elements: the Cgl1289M, UGI, pmCDA1 and TadA-ABE8e; the Cgl1289M, UGI, pmCDA1 and TadA-ABE8e are connected in series in sequence;
  • the MP5T includes the following elements: the temperature-sensitive replication origin site RepA101, the Cgl1289M, UGI, pmCDA1 and TadA-ABE8e; the RepA101, Cgl1289M, UGI, pmCDA1 and TadA-ABE8e are connected in series in sequence, and the core of the RepA101
  • the nucleotide sequence is shown in SEQ ID NO:53.
  • nucleotide sequence of RBS in the DNA polymerase Cgl1289M is shown in SEQ ID NO: 38.
  • the Cgl1289M and UGI share a promoter Ptac; the pmCDA1 and TadA-ABE8e share a promoter Psod.
  • the present invention also provides a nucleotide encoding the above mutant.
  • nucleotide sequences of MP1 to MP5 and MP5T are as shown in SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59 and SEQ ID NO:54. Show.
  • the present invention also provides an expression vector containing the above-mentioned mutant or nucleotide.
  • the expression vector includes any one of pXMJ19, pDXW10, and pEC-XK99E.
  • the present invention also provides a recombinant bacterium that expresses the above-mentioned mutant, nucleotide or expression vector.
  • the starting strain of the recombinant bacteria is Corynebacterium glutamicum 13032.
  • the invention also provides a high-efficiency mutation system of Corynebacterium glutamicum, which includes the above mutant and a mutagen.
  • the invention also provides a method for improving the mutagenesis efficiency of the above mutant, which includes the following steps: the mutant is induced at an inducer concentration of 0.5-1.5mM and an induction temperature of 25-35°C.
  • the invention also provides a method for constructing the above-mentioned recombinant bacteria, which includes the following steps: amplifying the nucleotide sequence of the mutant, connecting it to an expression vector, and then transforming the expression vector into Corynebacterium glutamicum to obtain the recombinant bacteria .
  • the invention also provides an application of the above-mentioned MP5T in preparing a plasmid elimination system.
  • the plasmid elimination system further includes a mutagen.
  • the MP5T achieves normal replication of the plasmid when the culture temperature is 25-35°C.
  • the culture temperature is 40-45°C, the plasmid cannot replicate normally, thereby eliminating the mutant plasmid.
  • the invention also provides an application of the above-mentioned mutant, nucleotide, expression vector or mutation system in screening high-producing glutamic acid chassis cells.
  • the invention also provides an application of the above mutant, nucleotide, expression vector or mutation system in the production of amino acids.
  • the invention also provides an application of the above mutant, nucleotide, expression vector or mutation system in screening acid-resistant strains.
  • the acid-tolerant strain is an acid-tolerant strain with high glutamic acid production.
  • the present invention has the following beneficial effects:
  • the present invention provides a high-efficiency mutant of Corynebacterium glutamicum.
  • the mutant of the present invention has a high mutation rate and a broad mutation spectrum.
  • the mutant MP5T also has the ability to achieve The elimination of the plasmid will not allow the mutant plasmid to remain in the cell and continue to cause background mutations, and also lays the foundation for subsequent metabolic engineering and genetic modification of the strain.
  • MP5T has a higher mutation rate than conventional mutagenesis methods, with a mutation frequency f as high as 1.66 ⁇ 10 -3 , that is to say , 1.66 positive mutants can be obtained from 1000 mutants.
  • the mutagenesis method of the mutant of the present invention is simple, and can continuously realize the accumulation of mutations in cells without causing any harm to the operator, greatly shortening the construction time of the mutant library, and avoiding the waste of resources and energy. Greatly improve work efficiency.
  • the present invention uses mutant screening to obtain acid-resistant mutant strains and improves the fermentation level of L-glutamic acid to a certain extent. Compared with normal laboratory evolutionary screening, the invention shortens the time for strain selection and also improves the fermentation level of L-glutamic acid to a certain extent. It provides acid-resistant chassis cells for subsequent systematic metabolic engineering to create glutamate-high-producing strains.
  • Figure 1 is a schematic diagram of the construction of a high-efficiency mutagenesis system for Corynebacterium glutamicum;
  • Figure 2 shows the homology comparison between DNA polymerase dnaQ926 and Cgl1289
  • Figure 3 shows the sequence alignment of the Exo I conserved region of DNA polymerases Cgl1289 and Cgl1243.
  • Figure 4 is the concentration curve of C.glutamicum 13032 resistant to rifampicin antibiotic
  • Figure 5 shows the cell survival of recombinant bacteria Cg-MP1 and Cg-MP-Cgl1289, in which the CK group is the control group without adding IPTG;
  • Figure 6 shows the mutation rate ⁇ bp and cell survival rate results of recombinant bacteria Cg-MP1, Cg-MP-Cgl1243M, Cg-MP-Cgl1289 and Cg-MP-Cgl1243;
  • A is the structure of MP1, MP2, MP3, MP4, and MP5 mutant plasmids
  • B is the cell survival rate under the influence of MP1, MP2, MP3, MP4, and MP5 mutant plasmids
  • C is the MP1, MP2, and MP5 mutant plasmids in C.glutamicum.
  • D is the cell survival of Cg-MP1, Cg-MP2, Cg-MP3, Cg-MP4 and Cg-MP5;
  • Figure 8 shows the determination of mutation spectrum of MPs mutant plasmids.
  • A shows the determination of mutation spectrum of MP1, MP3, MP4 and MP5;
  • B shows the determination of rpoB gene mutation frequency in MP1, MP3, MP4 and MP5;
  • Figure 9 shows the effect of different strength promoters on the mutation rate of mutant plasmid MPs
  • Figure 10 shows the effects of IPTG at different temperatures and concentrations on the mutation rate of mutant plasmid MP5-RBS6;
  • Figure 11 shows the elimination of mutant plasmid MP5T.
  • A shows the elimination of mutant plasmid MP5T on the BHI plate without chloramphenicol resistance
  • B shows the elimination of mutant plasmid MP5T on the BHI plate containing chloramphenicol resistance. Eliminate situations;
  • Figure 12 shows the comparison between MPT5 and other conventional mutagenesis techniques.
  • A is the frequency of rifampicin-resistant strains under different induction conditions;
  • B to C are the mutation spectra produced by ARTP, EMS and MNNG mutagenesis;
  • Figure 13 shows the screening of acid-resistant mutant strains.
  • A is the acid-resistant growth test result of a single colony on BHI (pH 5.3) solid plate;
  • B is the acid-resistant mutant strains NS-A1, NS-B4, NS-D1 and wild-type C.glutamicum 13032 Cell survival rate measurement results;
  • C is the cell survival of acid-resistant mutant strains NS-A1, NS-B4, NS-D1 and wild-type C.glutamicum 13032 cells measured by spot plate test on BHI plate;
  • Figure 14 shows the fermentation kinetic parameters of C.glutamicum 13032 and the acid-tolerant mutant strain NS-A1.
  • the invention provides a high-efficiency mutant of Corynebacterium glutamicum, which includes any one of MP1 to MP5 and MP5T:
  • the MP1 includes DNA polymerase Cgl1289M, and the amino acid sequence of the DNA polymerase Cgl1289M is shown in SEQ ID NO: 13;
  • the MP2 includes the following elements: the Cgl1289M and the uracil DNA glycosylase inhibitor UGI; the downstream tandem UGI of the Cgl1289M, and the amino acid sequence of the UGI is as shown in SEQ ID NO: 25;
  • the MP3 includes the following elements: the Cgl1289M, UGI and cytosine deaminase pmCDA1; the Cgl1289M, UGI and pmCDA1 are connected in series in sequence; the amino acid sequence of the pmCDA1 is as shown in SEQ ID NO: 27;
  • the MP4 includes the following elements: the Cgl1289M, UGI and adenine deaminase TadA-ABE8e; the Cgl1289M, UGI and TadA-ABE8e are connected in series in sequence, and the amino acid sequence of the TadA-ABE8e is as shown in SEQ ID NO: 29 .
  • the MP5 includes the following elements: the Cgl1289M, UGI, pmCDA1 and TadA-ABE8e; the Cgl1289M, UGI, pmCDA1 and TadA-ABE8e are connected in series in sequence;
  • the MP5T includes the following elements: the temperature-sensitive replication origin site RepA101, the Cgl1289M, UGI, pmCDA1 and TadA-ABE8e; the RepA101, Cgl1289M, UGI, pmCDA1 and TadA-ABE8e are connected in series in sequence, and the core of the RepA101
  • the nucleotide sequence is shown in SEQ ID NO:53.
  • the present invention through comparative analysis of DNA polymerases derived from C. glutamicum, it was found that the DNA polymerase Cgl1289 has a highly conserved Exo I region, and the included Exo I region has two highly conserved aspartic acid residues. Acid and glutamic acid residues, the present invention mutates two highly conserved aspartic acid and glutamic acid residues in the Exo I region of DNA polymerase Cgl1289 to alanine, that is, DNA polymerase Cgl1289M, The mutation rate and lethality rate of cells are significantly increased.
  • the nucleotide sequence of RBS in the DNA polymerase Cgl1289M is preferably as shown in SEQ ID NO: 38, which further improves the mutation rate of the mutant.
  • the Cgl1289M and UGI share a promoter Ptac; the pmCDA1 and TadA-ABE8e share a promoter Psod.
  • the present invention has tried to use the weakly inducible promoter Parac to express DNA polymerase Cgl1289M, but in Parac Under control, the mutation ability cannot be exerted, but the present invention directly selects the strong promoter Ptac to regulate the expression of DNA polymerase Cgl1289M, successfully enabling the mutant to exert its mutation function.
  • the selection of promoters to regulate the expression of PsodpmCDA1 and Tada-ABE8e is beneficial to improving the expression of the glutamic acid polymerase Cgl1289M. Mutation rate of Corynebacterium acidum.
  • the uracil DNA glycosylase inhibitor UGI is derived from Bacillus subtilis phage PBS2 and can disrupt the base excision repair system by inhibiting the activity of DNA glycosylase in C. glutamicum 13032 and can Increase mutation rate and decrease cell survival rate.
  • the cytosine deaminase pmCDA1 derived from the cytosine deaminase pmCDA1 of Petromyzon marinus can be expressed in C. glutamicum and can increase the mutation frequency of the base pair G-C to A-T; adenine deaminase TadA-ABE8e was evolved from E. coli by David Liu's team and can achieve adenine deamination in C. glutamicum using DNA as a substrate.
  • the expression of cytosine deaminase pmCDA1 and adenine deaminase TadA-ABE8e will significantly increase during DNA replication. mutation rate and broaden the base mutation spectrum.
  • MP5T includes the following elements: temperature-sensitive replication origin site RepA101, DNA polymerase Cgl1289, uracil DNA glycosylase inhibitor UGI, cytosine deaminase pmCDA1, adenine deaminase TadA- ABE8e has a synergistic effect between various components and can significantly increase the mutation rate of Corynebacterium glutamicum.
  • the above mutant of the present invention by measuring the mutation spectrum of the above mutant, it is found that the above mutant of the present invention not only has a high mutation rate, but also has a broad mutation spectrum.
  • the present invention also provides a nucleotide encoding the above mutant.
  • nucleotide sequences of MP1 to MP5 and MP5T are preferably such as SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59 and SEQ ID Shown in NO:54.
  • the present invention also provides an expression vector containing the above-mentioned mutant or nucleotide.
  • the expression vector preferably includes any one of pXMJ19, pDXW10, and pEC-XK99E, and is further preferably pXMJ19.
  • the present invention also provides a recombinant bacterium that expresses the above-mentioned mutant, nucleotide or expression vector.
  • the starting strain of the recombinant bacteria is preferably C. glutamicum 13032, purchased from Beina Biotech.
  • the invention also provides a method for constructing the above-mentioned recombinant bacteria, which includes the following steps: amplifying the nucleotide sequence of the mutant, connecting it to an expression vector, and then transforming the expression vector into Corynebacterium glutamicum to obtain the recombinant bacteria .
  • the invention also provides a high-efficiency mutation system of Corynebacterium glutamicum, which includes the above mutant and a mutagen.
  • the invention also provides a method for improving the mutagenesis efficiency of the above mutant, which includes the following steps: the mutant is induced at an inducer concentration of 0.5-1.5mM and an induction temperature of 25-35°C.
  • the mutant when the mutant is MP5, the inducer concentration is 1.0mM, and the induction temperature is 30°C, the mutagenesis efficiency reaches the highest level, and the mutation rate ⁇ bp reaches 6.12 ⁇ 10 -6 , which is higher than that of wild-type glutamine. Corynebacterium acidum increased 153,000 times.
  • the invention also provides an application of the above-mentioned MP5T in preparing a plasmid elimination system.
  • the plasmid elimination system also includes a mutagen.
  • the mutagen is preferably IPTG.
  • the MP5T achieves normal replication of the plasmid when the culture temperature is 25-35°C. When the culture temperature is 40-45°C, the plasmid cannot replicate normally, thereby eliminating the mutant plasmid.
  • the MP5T achieves normal replication of the plasmid when the culture temperature is 28-32°C. When the culture temperature is 41-43°C, the plasmid cannot replicate normally, thereby eliminating the mutant plasmid. .
  • the invention also provides an application of the above-mentioned mutant, nucleotide, expression vector or mutation system in screening high-producing glutamic acid chassis cells.
  • the invention also provides an application of the above mutant, nucleotide, expression vector or mutation system in the production of amino acids.
  • the amino acid preferably includes one or more of glutamic acid, valine, and lysine.
  • the invention also provides an application of the above mutant, nucleotide, expression vector or mutation system in screening acid-resistant strains.
  • the acid-tolerant strain is preferably an acid-tolerant strain with high glutamic acid production.
  • the acid-tolerant strain of the present invention can grow normally under the condition of pH ⁇ 5.3 and improves the production of L-glutamic acid.
  • LB medium used for E.coli culture: Yeast extract 5, Tryptone 10, NaCl 10, pH 7.0, sterilized at 121°C for 20 minutes, add to the required antibiotic concentration as needed. If you need to prepare a solid culture medium, you need to add 1.5 to 2% agar powder.
  • BHI medium (g/L) for C. glutamicum cell culture: BHI solid powder 38.5, pH 7.0, sterilized at 115°C for 20 minutes, add to the required antibiotic concentration as needed. If you need to prepare a solid culture medium, you need to add 1.5 to 2% agar powder.
  • BHIGS competent medium (g/L), used to prepare C. glutamicum competent cells: BHI solid powder 18.5, glycine 30, Tween 801, D-sorbitol 91, sterilized at 115°C for 20 minutes.
  • Fermentation seed medium (g/L): glucose 25, K 2 HPO 4 1.5, MgSO 4 0.6, corn steep liquor 30, FeSO 4 ⁇ 7H 2 O 0.005, MnSO 4 ⁇ H 2 00.005, urea 2.5 (sterilized separately), pH 7.3.
  • Fermentation medium g/L: Glucose 140, K 2 HPO 4 1, MgSO 4 0.6, corn steep liquor 5, FeSO 4 ⁇ 7H 2 O0.005, MnSO 4 ⁇ H 2 00.005, urea 7 (sterilized separately), pH 7.3.
  • ⁇ bp f/[R ⁇ ln(N/N 0 )].
  • ⁇ bp represents the base mutation rate per generation
  • the number of single clones, N 0 represents the number of single clones on the rifampicin-resistant plate when the growth of the single clone is just observed.
  • DNA polymerases Cgl1289 and Cgl1243 have a highly conserved Exo I region (the amino acid sequence of the DNA polymerase Cgl1289 is shown in SEQ ID NO: 1, as described The amino acid sequence of Cgl1243 is shown in SEQ ID NO:2), and the included Exo I region has two highly conserved aspartic acid and glutamic acid residues (see Figure 3).
  • DNA polymerase Cgl1289 and Cgl1243 gene fragments were amplified by PCR using the C. glutamicum 13032 genome as a template and Cgl1289-F/R and Cgl1243-F/R as primers respectively.
  • the pXMJ19 linearized vector was amplified by inverse PCR using the primer pair P19F/P19R.
  • the Cgl1289 and Cgl1243 gene fragments were connected to the linearized vector pXMJ19 through homologous recombination and transformed into the E.coli BL21 competent state. The correct positive transformants were identified by colony PCR and sent to a sequencing company for sequencing verification.
  • the verified recombinant vectors were MP-Cgl1289 and MP-Cgl1243. Then, the aspartic acid residues and glutamic acid residues of the two DNA polymerases Cgl1289 and Cgl1243 were mutated to alanine, and then the primer pairs Cgl1289M-F/R and Cgl1243M-F/R were used to express the vectors respectively.
  • MP-Cgl1289 and MP-Cgl1243 are template inverse PCR.
  • Recombinant vectors MP1 and MP-Cgl1243M were constructed according to the same method.
  • the recombinant plasmids MP1, MP-Cgl1243M, MP-Cgl1289 and MP-Cgl1243 were electroporated into C.glutamicum 13032 to construct recombinant bacteria Cg-MP1, Cg-MP-Cgl1243M, Cg-MP-Cgl1289 and Cg-MP-Cgl1243.
  • the primer sequences of Cgl1289-F/R, Cgl1243-F/R, P19F/P19R, Cgl1289M-F/R and Cgl1243M-F/R are as follows:
  • the amino acid sequence of the DNA polymerase Cgl1289M is shown in SEQ ID NO:13, and the nucleotide sequence of Cgl1289M is shown in SEQ ID NO:14; the amino acid sequence of the DNA polymerase Cgl1243M is shown in SEQ ID NO:15, The Cgl1243M nucleotide sequence is shown in SEQ ID NO:16.
  • the rpoB/Rif r system based on screening Rif r mutants has been successfully applied to the calculation of mutation rates of E.coli, C.glutamicum, etc. Therefore, in order to determine the mutation rates of recombinant strains Cg-MP1, Cg-MP-Cgl1243M, Cg-MP-Cgl1289 and Cg-MP-Cgl1243, the maximum inhibitory rate of rifampin resistance of wild-type C.glutamicum 13032 must first be determined. bacteria concentration. So C.
  • glutamicum 13032 cells cultured to the mid-logarithmic phase were taken and incubated in rifampicin-resistant solids containing 0 ⁇ g/mL, 0.5 ⁇ g/mL, 1 ⁇ g/mL, 1.5 ⁇ g/mL, 2 ⁇ g/mL, and 4 ⁇ g/mL.
  • the plates were streaked and 10-fold dilutions were spotted using sterile saline.
  • Wild-type C.glutamicum 13032, recombinant bacteria Cg-MP1, Cg-MP-Cgl1243M, Cg-MP-Cgl1289 and Cg-MP-Cgl1243 were cultured at 30°C and 180rpm. When in the mid-log phase, add 0.5 respectively. Expression was induced by mM IPTG, and the culture was continued for 24 to 36 hours at 30°C and 180 rpm. The cell culture was serially diluted 10 times and spread onto a solid BHI double antibody plate containing 15 ⁇ g/mL chloramphenicol and 10 ⁇ g/mL rifampicin. And the BHI solid plate containing only chloramphenicol resistance was cultured in a constant temperature incubator at 30°C for 24 hours.
  • plasmid MP6 purchased from Addgene
  • ugi-F/R as the primer
  • PCR amplification was performed to obtain the uracil DNA glycosylase inhibitor UGI gene fragment.
  • the uracil DNA glycosylase inhibitor UGI was expressed in series downstream of Cgl1289M to construct the recombinant vector MP2.
  • cytosine deaminase pmCDA1 and adenine deaminase were continued to be expressed in tandem downstream of UGI.
  • cytosine deaminase pmCDA1 and adenine deaminase TadA-ABE8e were continued to be expressed in tandem downstream of UGI, respectively.
  • the adenine deaminase TadA-ABE8e was continued to be serially expressed downstream of UGI, and the cytosine deaminase pmCDA1 and adenine deaminase TadA-ABE8e were serially expressed downstream of UGI to construct recombinant vectors MP3, MP4, and MP5 respectively.
  • Recombinant plasmids MP2, MP3, MP4, and MP5 were electroporated into C.
  • cytosine deaminase pmCDA1 and adenine deaminase TadA-ABE8e are regulated by promoter P sod .
  • the primer sequences of ugi-F/R, pmCDA1-F/R, tada-F/R and pmCDA1-tada F/R are as follows:
  • pmCDA1-F tacgaaaggatttttacccaaaggaggacaactaatgaccgacgcggaatacg(SEQ ID NO:19)
  • pmCDA1-tada F ccaaatctccggcggtttaaaaggaggacaactaatgtctgaagtggagttctccc(SEQ ID NO:23)
  • pmCDA1-tada R gctcggtacccggggatcctttaattgatggaggactgtgcct(SEQ ID NO:24)
  • the amino acid sequence of the uracil DNA glycosylase inhibitor UGI is shown in SEQ ID NO:25, and the nucleotide sequence of the UGI is shown in SEQ ID NO:26; the cytosine deaminase pmCDA1
  • the amino acid sequence is shown in SEQ ID NO:27, the nucleotide sequence of pmCDA1 is shown in SEQ ID NO:28; the amino acid sequence of the adenine deaminase TadA-ABE8e is shown in SEQ ID NO:29, The nucleotide sequence of TadA-ABE8e is shown in SEQ ID NO: 30.
  • the nucleotide sequence of MP1 is shown in SEQ ID NO:55; the nucleotide sequence of MP2 is shown in SEQ ID NO:56; the nucleotide sequence of MP3 is shown in SEQ ID NO:57
  • the nucleotide sequence of MP4 is shown in SEQ ID NO:58; the nucleotide sequence of MP5 is shown in SEQ ID NO:59.
  • the rifampicin resistance-encoding gene rpoB of the rifampicin-resistant mutant strains screened on the rifampicin-resistant plate after mutation was used for high-throughput sequencing to analyze the mutation spectrum.
  • MP5 expresses both cytosine deaminase pmCDA1 and adenine deaminase TadA-ABE8e, and is endowed with a broader base mutation distribution.
  • G:C-A:T, T:A-C The types of base mutations of G and A:T-G:C increased significantly (see Figure 8).
  • the rpoB mutation sites discovered based on sequencing basically include the reported mutation sites that can acquire rifampicin resistance.
  • Three new mutation sites were also discovered, such as g1241a, c1286g and c1313t, which can also make C .glutamicum acquires rifampicin resistance (Table 1).
  • the mutation sites marked underline represent nonsense mutations, and the mutation sites marked in bold font indicate that they are not included in the reported rpoB mutation library. They and the mutation sites are both reported to be able to obtain Rif r resistance. Sexual mutation.
  • a strong inducible promoter P tac
  • P tuf a strong constitutive promoter
  • P sod a medium-strength constitutive promoter
  • P zwf The constitutive promoter promoter drives the expression of cytosine deaminase pmCDA1 and adenine deaminase TadA-ABE8e.
  • the recombinant plasmid MP5 deleting the promoter P sod gene fragment was linearized by inverse PCR technology, and then using the C. glutamicum 13032 genome as a template to amplify the promoters P tuf and P zwf gene fragment, and then the promoter P tuf and P zwf gene fragment and the linearized MP5 plasmid were connected by homologous recombination, and transformed into E.coli BL21 competent cells to construct recombinant plasmids MPtuf and MPzwf respectively.
  • recombinant plasmids MPtac and MPdtac directly uses the recombinant plasmid MP5 as a template, designs corresponding primers and inverse PCR amplifies the corresponding linearized fragments, and then uses the homologous recombination ligation kit to sterilize the obtained linearized fragments at 37°C. Incubate for 30 minutes to allow self-ligation, and then transfer to E.coli BL21 competent cells to construct recombinant plasmids MPtac and MPdtac respectively.
  • the primer sequences of P5F/P5R are as follows:
  • the mutation rate was measured and found that the mutant plasmid MP5-RBS6 had the highest mutation rate, reaching 5.76 ⁇ 10 -6 , which was 1.5 times higher than MP5 (Table 4).
  • the MPs mutation plasmid needs to add the inducer IPTG to induce the expression of DNA polymerase Cgl1289M and DNA glycosylase inhibitor UGI. Therefore, the mutation rate of MPs will be affected by the concentration of the inducer and the induction temperature. Therefore, the effects of different concentrations of IPTG addition and induction temperature on the mutation rate of MP5-RBS6 mutant plasmid were examined.
  • MP5-RBS6 mutant plasmid induction method streak and activate C. glutamicum containing the MP5-RBS6 mutant plasmid on a plate containing chloramphenicol resistance, culture it upside down in a 30°C incubator for 24 to 48 hours, pick a single colony and inoculate it by adding chlorine 10mL BHI liquid culture medium containing chloramphenicol, cultured at 30°C, 180rpm for 24h, transferred to 50mL BHI liquid culture medium with chloramphenicol resistance according to 1% inoculation amount, continued culturing for 3h at 30°C, 180rpm, then added different amounts of IPTG, and placed Induced at different temperatures for 12h.
  • the mutagenesis efficiency is highest when the optimal induction temperature is 30°C, which is the optimal temperature for the growth of C. glutamicum (A and C in Figure 10). It may be because neither high temperature nor low temperature is conducive to the growth of C. glutamicum, and it will also hinder the normal replication of DNA, thus leading to a reduction in the mutation rate.
  • the induction temperature was controlled at 30°C.
  • the inducer concentration is 1mM and the induction temperature is 30°C
  • the mutagenesis efficiency of MP5-RBS6 plasmid reaches the highest level, and the mutation rate ⁇ bp reaches 6.12 ⁇ 10 -6 , which is higher than that of wild-type Corynebacterium glutamicum. 153,000 times.
  • the temperature-sensitive replication origin site RepA101 in the CRISPR system was used to replace the original replication origin site of MP5-RBS6 itself to construct the recombinant bacterium Cg-MP5T.
  • the specific construction process is as follows:
  • the RepA 101 gene fragment was amplified by PCR.
  • the RepA 101 gene fragment obtained by gel recovery and purification was combined with the deleted original plasmid replication origin site amplified by inverse PCR.
  • the linearized MP5 fragment was subjected to homologous recombination and ligation, and the ligation product was transformed into E.coli BL21 competent cells to construct the recombinant plasmid MP5T.
  • the recombinant plasmid MP5T was electroporated into C. glutamicum competent cells to construct the recombinant bacterium Cg-MP5T. in,
  • repA101-R atgtctgaattagttgttttcaaagcaaat(SEQ ID NO:52)
  • the nucleotide sequence of the temperature-sensitive replication origin site RepA101 is shown in SEQ ID NO:53; the nucleotide sequence of the MP5T is shown in SEQ ID NO:54.
  • the recombinant strain Cg-MP5T was streaked and activated on a BHI solid plate, and a single colony was picked and transferred to a BHI medium containing chloramphenicol resistance, and cultured at 30°C and 180 rpm for 18 to 24 hours. Then transfer to anti-antibody BHI medium, continue culturing for 12 hours at 42°C and 180rpm, streak on the anti-antibody BHI plate, and continue culturing in a 30°C constant-temperature incubator for 24 to 36 hours.
  • the single colonies growing on the non-anti-BHI plate were placed on the non-anti-BHI culture plate and the BHI culture plate containing chloramphenicol in one-to-one correspondence, and the culture was continued in a 30°C incubator for 18 to 24 hours.
  • the strains with corresponding numbers that do not grow on the chloramphenicol plate and grow on the antibiotic-free plate are the strains whose MP5T plasmid has been successfully lost.
  • MP5T can replicate normally at 30°C, and can easily eliminate plasmids at 42°C (see Figure 11).
  • the successful construction of MP5T enables the MP5 mutant plasmid not only to have a high mutation rate and a broad mutation spectrum, but also to eliminate the plasmid after completing the screening of mutant strains, so that the mutant plasmid will not continue to remain in the cell.
  • Continuously causing background mutations also lays the foundation for subsequent metabolic engineering and genetic modification of strains.
  • Traditional mutagenesis methods mainly include physical and chemical mutagenesis methods.
  • the effects of four mutagenesis methods, EMS, MNNG, UV and ARTP, in C. glutamicum were further analyzed. mutation effect.
  • UV mutagenesis (3) Ultraviolet (UV) mutagenesis:
  • the cells obtained by the above mutagenesis method were cultured and spread on 10-fold gradient dilutions on anti-BHI solid plates and rifampicin antibiotic-resistant plates containing a final concentration of 10 ⁇ g/mL, and placed upside down in a 30°C constant-temperature incubator. After static culture for 24 to 36 hours, count the number of colonies growing on each rifampicin-resistant BHI plate and non-anti-BHI plate, and calculate the mutation rate.
  • MP5T has a higher mutation rate than the other four mutagenesis methods, with a mutation frequency f as high as 1.66 ⁇ 10 -3 , that is to say, 1.66 positive mutants can be obtained out of 1000 mutants.
  • the mutation frequency of chemical mutagens EMS and MNNG in C. glutamicum is not very high.
  • chemical mutagens such as EMS and MNNG are strong carcinogens and are difficult to degrade in the environment. They have persistent toxic effects on the environment and human body, and have certain potential dangers.
  • ARTP mutagenesis has a relatively broad mutation spectrum (see Figure 12), its mutation rate is not much different from that of the chemical mutagens EMS and MNNG, and the mutation rate in C. glutamicum is still low.
  • the process of screening rifampicin-resistant mutant strains using ultraviolet mutagenesis we basically did not obtain positive mutant strains. This may be due to the low efficiency of ultraviolet mutagenesis and the sequence preference of mutation direction, which is mainly concentrated in C:GA. :T mutation.
  • UV and ARTP mutagenesis are relatively safe and reliable compared with chemical mutagens, UV and ARTP mutagenesis usually constitute a single round of mutagenesis and cannot achieve continuous mutagenesis like chemical mutagens.
  • MP5T mutation plasmid not only has a high mutation rate, wide mutation spectrum, and simple operation method, but also can continuously accumulate mutations in cells without causing any harm to the operator, greatly shortening the construction time of mutant library , avoiding the waste of resources and energy, and greatly improving work efficiency.
  • the NS-A1 strain was fermented in a 5L fermenter.
  • the specific operation was as follows: take the NS-A1 preserved strain frozen tube, draw lines on the BHI solid petri dish, and isolate single colonies. Then use an inoculating loop to pick a single colony in BHI liquid culture medium and insert it into it, and culture it at 30°C and 180 rpm for 12 hours to activate the cells. Then, the activated cells (inoculation amount 1%) were inserted into 10 mL of fermentation seed medium, cultured at 30°C and 180 rpm for 12 hours, and then transferred to 200 mL of seed medium to continue culturing for 16-18 hours.

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Abstract

本发明提供了一种谷氨酸棒杆菌高效突变体及重组菌构建方法与应用，属于基因工程和诱变技术领域，基于筛选来源于C.glutamicum自身DNA复制过程中的DNA聚合酶，得到高度保守性的Exo I区域，并通过定点突变解除DNA聚合酶的校对、修复功能，提高突变率。基于筛选得到的DNA聚合酶突变体，逐步串联组装尿嘧啶DNA糖基化酶抑制剂UGI、胞嘧啶脱氨酶pmCDA1和腺嘌呤脱氨酶TadA-ABE8e，构建一系列突变质粒MPs，该突变体在C.glutamicum中实现高效的突变率以及广泛的突变谱，且突变过程绿色、健康，没有有毒有害的物质参与，本发明构建的谷氨酸棒杆菌高效突变体系，可用于筛选高产谷氨酸底盘细胞，也为应用于其他氨基酸生产提供了重要借鉴。

Description

一种谷氨酸棒杆菌高效突变体及重组菌构建方法与应用 技术领域
本发明属于基因工程和诱变技术领域，尤其涉及一种谷氨酸棒杆菌高效突变体及重组菌构建方法与应用。
背景技术
目前针对谷氨酸棒杆菌(C.glutamicum)的突变方法还是比较传统的诱变方法，如紫外诱变、ARTP诱变、化学诱变等，比较费时费力，且突变频率不高。紫外诱变能够造成基因组随机突变，操作简单，但是有一定的序列偏好性，突变效率较低，且诱变的过程中的紫外线容易对实验人员的皮肤产生损伤。相对于紫外诱变，ARTP诱变具有更高的突变率和更广的突变谱，且操作简单，但是ARTP诱变需要消耗大量的活性粒子(如氦粒子)，耗费能量。化学诱变剂如甲基磺酸乙酯和1-甲基-3-硝基-1-亚硝基胍，可以用来实现突变体文库的构建以及菌株选育，但是化学诱变产生的突变谱比较狭窄，而且使用的化学突变试剂通常都是剧毒性物质、强有力的人类致癌物。碱基突变类型一般具有12种，通过更多的覆盖12种突变类型，能够赋予突变子所获得的的氨基酸具有更多的突变方向，也就说更广的突变谱能够促进进化的遗传多样性。常规的实验室进化突变方法都存在一定的弊端，因此，寻求突变条件更加温和、环保、健康和安全以及具备更高突变效率和突变谱的随机突变体系引发了人们的广泛关注。
哈佛大学David Liu团队开发了一种全新的突变质粒MP6，在阿拉伯糖诱导的PBAD启动子的调控下，表达了DNA聚合酶DnaQ926、DNA甲基化酶(DAM)、半甲基化GATC结合域(SeqA)、尿嘧啶DNA糖基化酶抑制剂UGI、胞嘧啶脱氨酶(CdaI)和细胞内dNTP池转录抑制因子(EmrR)，导致染色体突变率比野生型大肠杆菌提高了322,000倍。相对于XL1-Red和EndoMS/NucS突变系统，MP6突变质粒具有更高的突变率以及更广的突变谱，突变率高达μbp＝6.2×10 -6。
大肠杆菌体系虽然具备了高效突变体系MP6，但是实验室前期研究发现，MP6突变质粒基本不能够在谷氨酸棒杆菌发挥其高效的诱变能力，因此，构建一种谷氨酸棒杆菌突变体系并将其应用于氨基酸生产中是亟待解决的技术问题。
发明内容
为了克服现有技术的上述缺点，本发明提供一种谷氨酸棒杆菌高效突变体及重组菌构建方法与应用。
为了解决上述技术问题，本发明采用的技术方案是：
本发明提供了一种谷氨酸棒杆菌高效突变体，所述突变体包括MP1～MP5和MP5T中的任意一种：
所述MP1包括DNA聚合酶Cgl1289M，所述DNA聚合酶Cgl1289M的氨基酸序列如SEQ ID NO:13所示；
所述MP2包括如下元件：所述Cgl1289M和尿嘧啶DNA糖基化酶抑制剂UGI；所述Cgl1289M的下游串联UGI，所述UGI的氨基酸序列如SEQ ID NO:25所示；
所述MP3包括如下元件：所述Cgl1289M、UGI和胞嘧啶脱氨酶pmCDA1；所述Cgl1289M、UGI和pmCDA1依次串联；所述pmCDA1的氨基酸序列如SEQ ID NO:27所示；
所述MP4包括如下元件：所述Cgl1289M、UGI和腺嘌呤脱氨酶TadA-ABE8e；所述Cgl1289M、UGI和TadA-ABE8e依次串联，所述TadA-ABE8e的氨基酸序列如SEQ ID NO:29所示。
所述MP5包括如下元件：所述Cgl1289M、UGI、pmCDA1和TadA-ABE8e；所述Cgl1289M、UGI、pmCDA1和TadA-ABE8e依次串联；
所述MP5T包括如下元件：温度敏感型复制起始位点RepA101、所述Cgl1289M、UGI、pmCDA1和TadA-ABE8e；所述RepA101、Cgl1289M、UGI、pmCDA1和TadA-ABE8e依次串联，所述RepA101的核苷酸序列如SEQ ID NO:53所示。
优选地，所述DNA聚合酶Cgl1289M中RBS的核苷酸序列如SEQ ID NO:38所示。
优选地，所述Cgl1289M和UGI共用一个启动子Ptac；所述pmCDA1和TadA-ABE8e共用一个启动子Psod。
本发明还提供了一种编码上述突变体的核苷酸。
优选地，MP1～MP5和MP5T的核苷酸序列如SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59和SEQ ID NO:54所示。
本发明还提供了一种含有上述所述的突变体或核苷酸的表达载体。
优选地，所述表达载体包括pXMJ19、pDXW10、pEC-XK99E中的任意一种。
本发明还提供了一种重组菌，所述重组菌表达上述的突变体、核苷酸或表达载体。
优选地，所述重组菌的出发菌株为谷氨酸棒杆菌13032。
本发明还提供了一种谷氨酸棒杆菌高效突变体系，所述突变体系包括上述突变体和诱变剂。
本发明还提供了一种提高上述突变体诱变效率的方法，包括如下步骤：所述突变体在诱导剂浓度为0.5～1.5mM，诱导温度为25～35℃下诱导。
本发明还提供了一种上述重组菌的构建方法，包括如下步骤：扩增突变体的核苷酸序列，连接到表达载体上，然后将表达载体转化到谷氨酸棒杆菌中，得到重组菌。
本发明还提供了一种上述MP5T在制备质粒消除体系中的应用。
优选地，所述质粒消除体系还包括诱变剂。
优选地，所述MP5T在培养温度为25～35℃条件下实现质粒的正常复制，当培养温度为40～45℃时，质粒无法正常复制，从而实现突变质粒的消除。
本发明还提供了一种上述突变体、核苷酸、表达载体或突变体系在筛选高产谷氨酸底盘细胞中的应用。
本发明还提供了一种上述突变体、核苷酸、表达载体或突变体系在生产氨基酸中的应用。
本发明还提供了一种上述突变体、核苷酸、表达载体或突变体系在筛选耐酸型菌株中的应用。
优选地，所述耐酸性菌株为高产谷氨酸耐酸性菌株。
相对于现有技术，本发明具有如下有益效果：
(1)本发明提供了一种谷氨酸棒杆菌高效突变体，本发明的突变体具备了高效 的突变率以及广阔的突变谱，尤其是突变体MP5T还具备了完成突变株筛选后，实现质粒的消除，不会使得突变质粒继续留存在细胞内持续造成本底突变，也为后续菌株的代谢工程改造以及遗传改造奠定了基础。
(2)与常规突变方法(如化学诱变、紫外诱变、ARTP诱变)相比，MP5T比常规诱变方法具有更高的突变率，突变频率f高达1.66×10 -3，也就是说，1000个突变子中就能得到1.66个阳性突变子。而且，本发明的突变体的诱变方法简单，而且可以使持续在细胞内实现突变的积累而不对操作者造成任何伤害，极大的缩短突变株文库构建时间，避免了资源和能源的浪费，大大提高了工作效率。
(3)本发明利用突变体筛选得到耐酸型突变菌株且在一定程度上提高了L-谷氨酸的发酵水平，相对于正常的实验室进化筛选，缩短了菌种选育的时间，同时也为后续系统代谢工程改造打造谷氨酸高产菌株提供了耐酸型底盘细胞。
说明书附图
图1为谷氨酸棒杆菌高效诱变体系的构建示意图；
图2为DNA聚合酶dnaQ926与Cgl1289同源性比对；
图3为DNA聚合酶Cgl1289和Cgl1243的Exo I保守区域序列比对
图4为C.glutamicum 13032耐受利福平抗生素浓度曲线；
图5为重组菌Cg-MP1、Cg-MP-Cgl1289的细胞存活情况，其中CK组为不添加IPTG的对照组；
图6为重组菌Cg-MP1、Cg-MP-Cgl1243M、Cg-MP-Cgl1289和Cg-MP-Cgl1243的突变率μ bp以及细胞存活率结果图；
图7中A为MP1、MP2、MP3、MP4、MP5突变质粒的结构；B为MP1、MP2、MP3、MP4、MP5突变质粒的作用下的细胞存活率；C为C.glutamicum中MP1、MP2、MP3、MP4、MP5的突变率μ bp；D为Cg-MP1、Cg-MP2、Cg-MP3、Cg-MP4和Cg-MP5的细胞存活情况；
图8为MPs突变质粒突变谱的测定，A为MP1、MP3、MP4和MP5突变谱测定；B为MP1、MP3、MP4和MP5中rpoB基因突变频率测定；
图9为不同强度启动子对突变质粒MPs突变率的影响；
图10为不同温度和不同浓度的IPTG对突变质粒MP5-RBS6突变率的影响；
图11为突变体质粒MP5T的消除，A为不含氯霉素抗性的BHI平板上的突变体质粒MP5T的消除情况；B为含有氯霉素抗性的BHI平板上的突变体质粒MP5T的消除情况；
图12为MPT5与其他常规诱变技术的比较，A为不同诱导条件下耐受利福平抗性菌株出现的频率；B～C为ARTP、EMS和MNNG诱变产生的突变谱图；
图13为耐酸突变株的筛选，A为BHI(pH 5.3)固体平板上单菌落耐酸性生长检测结果；B为耐酸突变株NS-A1、NS-B4、NS-D1和野生型C.glutamicum 13032细胞存活率测定结果；C为耐酸突变株NS-A1、NS-B4、NS-D1和野生型C.glutamicum 13032细胞在BHI平板进行点板试验测得的细胞存活情况；
图14为C.glutamicum 13032和耐酸突变株NS-A1的发酵动力学参数。
具体实施方式
下面结合实施例和附图对本发明进一步说明。
本发明提供了一种谷氨酸棒杆菌高效突变体，所述突变体包括MP1～MP5和MP5T中的任意一种：
所述MP1包括DNA聚合酶Cgl1289M，所述DNA聚合酶Cgl1289M的氨基酸序列如SEQ ID NO:13所示；
所述MP2包括如下元件：所述Cgl1289M和尿嘧啶DNA糖基化酶抑制剂UGI；所述Cgl1289M的下游串联UGI，所述UGI的氨基酸序列如SEQ ID NO:25所示；
所述MP3包括如下元件：所述Cgl1289M、UGI和胞嘧啶脱氨酶pmCDA1；所述Cgl1289M、UGI和pmCDA1依次串联；所述pmCDA1的氨基酸序列如SEQ ID NO:27所示；
所述MP4包括如下元件：所述Cgl1289M、UGI和腺嘌呤脱氨酶TadA-ABE8e；所述Cgl1289M、UGI和TadA-ABE8e依次串联，所述TadA-ABE8e的氨基酸序列如SEQ ID NO:29所示。
所述MP5包括如下元件：所述Cgl1289M、UGI、pmCDA1和TadA-ABE8e；所述Cgl1289M、UGI、pmCDA1和TadA-ABE8e依次串联；
所述MP5T包括如下元件：温度敏感型复制起始位点RepA101、所述Cgl1289M、UGI、pmCDA1和TadA-ABE8e；所述RepA101、Cgl1289M、UGI、pmCDA1和TadA-ABE8e依次串联，所述RepA101的核苷酸序列如SEQ ID NO:53所示。
在本发明中，通过对C.glutamicum来源的DNA聚合酶进行比对分析，发现DNA聚合酶Cgl1289具有高度保守的Exo I区域，且所包含的Exo I区域中具有两个高度保守的天冬氨酸和谷氨酸残基，本发明把DNA聚合酶Cgl1289的Exo I区域中的两个高度保守的天冬氨酸和谷氨酸残基都突变为丙氨酸之后，即DNA聚合酶Cgl1289M，使得细胞的突变率和致死率显著提高。
在本发明中，所述DNA聚合酶Cgl1289M中RBS的核苷酸序列优选地如SEQ ID NO:38所示，进一步提高了突变体的突变率。
在本发明中，所述Cgl1289M和UGI共用一个启动子Ptac；所述pmCDA1和TadA-ABE8e共用一个启动子Psod，本发明曾尝试利用弱诱导型启动子Parac来表达DNA聚合酶Cgl1289M，但在Parac调控下，并不能发挥突变能力，但本发明直接选择强启动子Ptac调控DNA聚合酶Cgl1289M的表达，成功使得突变体发挥了突变功能，同时选择启动子调控PsodpmCDA1和Tada-ABE8e表达有利于提高谷氨酸棒杆菌的突变率。
在本发明中，所述尿嘧啶DNA糖基化酶抑制剂UGI来源于枯草芽孢杆菌噬菌体PBS2，能够在C.glutamicum 13032中通过抑制DNA糖基化酶的活性，扰乱碱基切除修复系统并且能够提高突变率以及降低细胞存活率。
在本发明中，所述胞嘧啶脱氨酶pmCDA1来源于Petromyzon marinus的胞嘧啶脱氨酶pmCDA1可在C.glutamicum中表达，并能增加碱基对G-C到A-T的突变频率；腺嘌呤脱氨酶TadA-ABE8e为David Liu团队从大肠杆菌中进化而来，能够在C.glutamicum中以DNA为底物实现腺嘌呤脱氨基。
在本发明中，当碱基切除修复系统即UGI和DNA聚合酶丧失校对核酸外切酶活 性时，胞嘧啶脱氨酶pmCDA1和腺嘌呤脱氨酶TadA-ABE8e的表达会显著增加DNA复制过程中的突变率以及拓宽碱基突变谱。
在本发明中，MP5T包括如下元件：温度敏感型复制起始位点RepA101、DNA聚合酶Cgl1289、尿嘧啶DNA糖基化酶抑制剂UGI、胞嘧啶脱氨酶pmCDA1、腺嘌呤脱氨酶TadA-ABE8e，各元件之间具有协同增效的作用，可显著提高谷氨酸棒杆菌的突变率。
在本发明中，通过对上述突变体的突变谱测定，本发明的上述突变体不仅拥有高的突变率，同时也具备广泛的突变谱。
本发明还提供了一种编码上述突变体的核苷酸。
在本发明中，MP1～MP5和MP5T的核苷酸序列优选地如SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59和SEQ ID NO:54所示。
本发明还提供了一种含有上述所述的突变体或核苷酸的表达载体。
在本发明中，所述表达载体优选地包括pXMJ19、pDXW10、pEC-XK99E中的任意一种，进一步优选地为pXMJ19。
本发明还提供了一种重组菌，所述重组菌表达上述的突变体、核苷酸或表达载体。
在本发明中，所述重组菌的出发菌株优选为C.glutamicum 13032，购自北纳生物。
本发明还提供了一种上述重组菌的构建方法，包括如下步骤：扩增突变体的核苷酸序列，连接到表达载体上，然后将表达载体转化到谷氨酸棒杆菌中，得到重组菌。
本发明还提供了一种谷氨酸棒杆菌高效突变体系，所述突变体系包括上述突变体和诱变剂。
本发明还提供了一种提高上述突变体诱变效率的方法，包括如下步骤：所述突变体在诱导剂浓度为0.5～1.5mM，诱导温度为25～35℃下诱导。
在本发明中，当所述突变体为MP5，诱导剂浓度为1.0mM，诱导温度为30℃时，诱变效率达到最高，突变率μbp达到了6.12×10 -6，相对于野生型谷氨酸棒杆菌提高了153000倍。
本发明还提供了一种上述MP5T在制备质粒消除体系中的应用。
在本发明中，所述质粒消除体系还包括诱变剂。在本发明中，所述诱变剂优选为IPTG。所述MP5T在培养温度为25～35℃条件下实现质粒的正常复制，当培养温度为40～45℃时，质粒无法正常复制，从而实现突变质粒的消除。在本发明一优选的实施方式中，所述MP5T在培养温度为28～32℃条件下实现质粒的正常复制，当培养温度为41～43℃时，质粒无法正常复制，从而实现突变质粒的消除。
本发明还提供了一种上述突变体、核苷酸、表达载体或突变体系在筛选高产谷氨酸底盘细胞中的应用。
本发明还提供了一种上述突变体、核苷酸、表达载体或突变体系在生产氨基酸中的应用。
在本发明中，所述氨基酸优选地包括谷氨酸、缬氨酸、赖氨酸中的一种或多种。
本发明还提供了一种上述突变体、核苷酸、表达载体或突变体系在筛选耐酸型菌株中的应用。
在本发明中，所述耐酸性菌株优选为高产谷氨酸耐酸性菌株。在本发明中，本发明的耐酸型菌株能够在pH≤5.3条件下正常生长，且提高了L-谷氨酸的产量。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
下述实施例中涉及的培养基如下：
LB培养基(g/L)，用于E.coli培养：Yeast extract 5，Tryptone 10，NaCl 10，pH 7.0，121℃灭菌20min，根据需要添加至所需要的抗生素浓度。如需配置固体培养基，则需要加入1.5～2％的琼脂粉。
BHI培养基(g/L)，用于C.glutamicum细胞培养：BHI固体粉末38.5，pH 7.0，115℃灭菌20min，根据需要添加至所需抗生素浓度。如需配置固体培养基，则需要加入1.5～2％的琼脂粉。
BHIGS感受态培养基(g/L)，用于制备C.glutamicum感受态细胞：BHI固体粉末18.5，甘氨酸30，Tween 801，D-山梨醇91，115℃灭菌20min。
发酵种子培养基(g/L)：葡萄糖25，K 2HPO 41.5,MgSO 40.6，玉米浆30，FeSO 4·7H 2O 0.005，MnSO 4·H 200.005，尿素2.5(单独灭菌)，pH 7.3。
发酵培养基(g/L)：葡萄糖140,K 2HPO 41，MgSO 40.6，玉米浆5，FeSO 4·7H 2O0.005，MnSO 4·H 200.005，尿素7(单独灭菌)，pH 7.3。
补料培养基(g/L)：葡萄糖800。
下述实施例中涉及的突变率及致死率计算方法如下：
碱基突变率计算公式：
μ bp＝f/[R×ln(N/N 0)]。μ bp表示每代碱基突变率，f表示利福平耐受突变体频率(f＝P/P 0，P表示添加IPTG诱导后在氯霉素和利福平抗生素双抗平板上的菌落数，P 0表示添加IPTG诱导后在氯霉素单抗平板上生长的菌落数)，R代表产生利福平耐药性特殊位点的突变个数，N表示利福平抗性平板上最终的单克隆个数，N 0表示刚观察到单克隆生长时的利福平抗性平板上的单克隆个数。
致死率计算公式：
μ＝M/M 0，其中添加IPTG诱导的氯霉素平板上的菌落数为M，未添加IPTG诱导的氯霉素平板上的菌落数M 0。
实施例1
1.1 C.glutamicum内源性DNA聚合酶的筛选
大肠杆菌体系虽然具备了高效的突变体系MP6，但研究发现，MP6突变质粒在C.glutamicum 13032中基本不能够发挥其高效的诱变能力。影响MP6突变质粒的关键酶是DNA聚合酶dnaQ926，通过将DNA聚合酶dnaQ926与C.glutamicum内源性的DNA聚合酶进行序列比对分析，发现同源性最高的DNA聚合酶为Cgl1289，且同源性只有18.1％(见图2)。猜测可能是danQ926的低序列同源性，限制了MP6在C.glutamicum 13032中的突变率。因此，寻找C.glutamicum内源性的DNA聚合酶，为在C.glutamicum中构建高效突变体系构建基础。
通过对C.glutamicum来源的DNA聚合酶进行比对分析，发现DNA聚合酶Cgl1289和Cgl1243具有高度保守的Exo I区域(所述DNA聚合酶Cgl1289的氨基酸序列如SEQ ID NO:1所示，所述Cgl1243的氨基酸序列如SEQ ID NO:2所示)，且所包含的Exo I区域中具有两个高度保守的天冬氨酸和谷氨酸残基(见图3)。
本实施例研究表明，当把Exo I区域中的两个高度保守的天冬氨酸和谷氨酸残基都突变为丙氨酸之后，会使得细胞的突变率和致死率显著提高。因此，分别将Cgl1289和Cgl1243两个DNA聚合酶的天冬氨酸残基和谷氨酸残基都突变为丙氨酸，具体流程如下：
以C.glutamicum 13032基因组为模板，分别以Cgl1289-F/R和Cgl1243-F/R为引物PCR扩增出DNA聚合酶Cgl1289和Cgl1243基因片段。以pXMJ19质粒为模板，利用引物对P19F/P19R反向PCR扩增pXMJ19线性化载体。而后将Cgl1289和Cgl1243基因片段分别与线性化载体pXMJ19进行同源重组连接，化转至E.coli BL21感受态。菌落PCR鉴定正确的阳性转化子并送测序公司测序验证，比对测序得到的结果，验证无误的重组载体即为MP-Cgl1289和MP-Cgl1243。而后将Cgl1289和Cgl1243两个DNA聚合酶的天冬氨酸残基和谷氨酸残基都突变为丙氨酸，再分别利用引物对Cgl1289M-F/R和Cgl1243M-F/R分别以表达载体MP-Cgl1289和MP-Cgl1243为模板反向PCR。按相同方法构建获得重组载体MP1和MP-Cgl1243M。将重组质粒MP1、MP-Cgl1243M、MP-Cgl1289和MP-Cgl1243电转化转化到C.glutamicum 13032中，构建重组菌Cg-MP1、Cg-MP-Cgl1243M、Cg-MP-Cgl1289和Cg-MP-Cgl1243。其中，Cgl1289-F/R、Cgl1243-F/R、P19F/P19R、Cgl1289M-F/R和Cgl1243M-F/R引物序列具体如下：
Cgl1289-F：aaacagaattaattaagcttaaaggaggacaactagtgttggggcgtcgaaaag(SEQ ID NO:3)
Cgl1289-R：gagtcgacctgcaggcatgcctattcccaaatatctttgagggtgttcg(SEQ ID NO:4)
Cgl1243-F：aaacagaattaattaagcttaaaggaggacaactaatgaactcaccaagcaatccat(SEQ ID NO:5)
Cgl1243-R：gagtcgacctgcaggcatgcttaggatgcggggtcagtt(SEQ ID NO:6)
P19-F：gcatgcctgcaggtcgac(SEQ ID NO:7)
P19-R：aagcttaattaattctgtttcctgtgtga(SEQ ID NO:8)
Cgl1289M-F：ctgtggccgtggcaacgac(SEQ ID NO:9)
Cgl1289M-R：gtcgttgccacggccacag(SEQ ID NO:10)
Cgl1243M-F：ctccttcgccctggcaacaa(SEQ ID NO:11)
Cgl1243M-R：ttgttgccagggcgaaggag(SEQ ID NO:12)
所述DNA聚合酶Cgl1289M的氨基酸序列如SEQ ID NO:13所示，Cgl1289M核苷酸序列如SEQ ID NO:14所示；所述DNA聚合酶Cgl1243M的氨基酸序列如SEQ ID NO:15所示，Cgl1243M核苷酸序列如SEQ ID NO:16所示。
基于筛选Rif r突变体的rpoB/Rif r系统已经成功应用于E.coli、C.glutamicum等的突变率的计算。因此，为了测定重组菌Cg-MP1、Cg-MP-Cgl1243M、Cg-MP-Cgl1289和Cg-MP-Cgl1243的突变率，首先要测定野生型C.glutamicum 13032耐受利福平抗性的最大抑菌浓度。于是取培养至对数中期的C.glutamicum 13032细胞，并分别在 含有0μg/mL、0.5μg/mL、1μg/mL、1.5μg/mL、2μg/mL和4μg/mL的利福平抗性固体平板上划线以及利用灭菌的生理盐水进行10倍稀释点板。
计算细胞存活率发现，当添加利福平抗性浓度为2μg/mL时，C.glutamicum 13032的细胞存活率仅为1.35％，当添加利福平抗性浓度为4μg/mL时，C.glutamicum 13032的细胞基本不能存活(见图4)。因此，后续试验测定突变率所选取的利福平浓度为10μg/mL，以保证只有获得了利福平抗性突变菌株方能存活。
将野生型C.glutamicum 13032、重组菌Cg-MP1、Cg-MP-Cgl1243M、Cg-MP-Cgl1289和Cg-MP-Cgl1243在30℃，180rpm条件下培养，当处于对数中期时，分别添加0.5mM IPTG诱导表达，继续在30℃，180rpm条件下培养24～36h，将细胞培养物10倍梯度稀释并涂布到含有15μg/mL氯霉素和10μg/mL利福平的固体BHI双抗平板和只含有氯霉素抗性的BHI固体平板，并于30℃恒温培养箱静置培养24h。
结果表明，重组菌Cg-MP1的细胞存活率仅有82.4％，突变率μ bp达到了1.2×10 -7，相对于野生型对照菌株C.glutamicum 13032提高了3000倍，重组菌Cg-Cgl1289由于没有将Exo I区域中的天冬氨酸残基和谷氨酸残基突变为丙氨酸，所以细胞存活率和突变率基本变化不大，但是重组菌Cg-1243M并没有像预期一样具有很高的突变率(见图5和图6)。说明Cgl1289的Exo I区域中的天冬氨酸残基和谷氨酸残基突变为丙氨酸是显著提高突变率的关键。
1.2突变体元器件筛选组装
以质粒MP6(购自Addgene)为模板，以ugi-F/R为引物，PCR扩增获得尿嘧啶DNA糖基化酶抑制剂UGI基因片段。在MP1基础上，在Cgl1289M的下游串联表达尿嘧啶DNA糖基化酶抑制剂UGI，构建重组载体MP2。分别以质粒MP6和苏州金唯智生物有限公司合成的质粒pET28a-TadA-ABE8e为模板，pmCDA1-F/R和tada-F/R为引物PCR分别扩增获得胞嘧啶脱氨酶pmCDA1和腺嘌呤脱氨酶TadA-ABE8e基因片段，pmCDA1-tada F/R扩增获得胞嘧啶脱氨酶pmCDA1和腺嘌呤脱氨酶TadA-ABE8e串联的基因片段。而后在MP2的基础上，在UGI的下游继续串联表达胞嘧啶脱氨酶pmCDA1和腺嘌呤脱氨酶TadA-ABE8e，具体地，分别在UGI的下游继续串联表达胞嘧啶脱氨酶pmCDA1、在UGI的下游继续串联腺嘌呤脱氨酶TadA-ABE8e、在UGI的下游继续串联表达胞嘧啶脱氨酶pmCDA1和腺嘌呤脱氨酶TadA-ABE8e，分别构建重组载体MP3、MP4、MP5。重组质粒MP2、MP3、MP4、MP5分别电转化到C.glutamicum 13032中构建获得重组菌Cg-MP2、Cg-MP3、Cg-MP4和Cg-MP5。其中胞嘧啶脱氨酶pmCDA1和腺嘌呤脱氨酶TadA-ABE8e受到启动子P sod所调控。其中，ugi-F/R、pmCDA1-F/R、tada-F/R和pmCDA1-tada F/R引物序列具体如下：
ugi-F：aaaattaggaggaatttcaacatgacaaatttatc(SEQ ID NO:17)
ugi-R：gctcggtacccggggatcctttataacattttaattttattttctccattactgtct(SEQ ID NO:18)
pmCDA1-F：tacgaaaggattttttacccaaaggaggacaactaatgaccgacgcggaatacg(SEQ ID NO:19)
pmCDA1-R：gctcggtacccggggatcctttaaaccgccggagatttg(SEQ ID NO:20)
tada-F：tacgaaaggattttttacccaaaggaggacaactaatgtctgaagtggagttctccc(SEQ ID NO:21)
tada-R：gctcggtacccggggatcctttaattgatggaggactgtgcct(SEQ ID NO:22)
pmCDA1-tada F：ccaaatctccggcggtttaaaaaggaggacaactaatgtctgaagtggagttctccc(SEQ ID NO:23)
pmCDA1-tada R：gctcggtacccggggatcctttaattgatggaggactgtgcct(SEQ ID NO:24)
所述尿嘧啶DNA糖基化酶抑制剂UGI的氨基酸序列如SEQ ID NO:25所示，所述UGI的核苷酸序列如SEQ ID NO:26所示；所述胞嘧啶脱氨酶pmCDA1的氨基酸序列如SEQ ID NO:27所示，所述pmCDA1的核苷酸序列如SEQ ID NO:28所示；所述腺嘌呤脱氨酶TadA-ABE8e的氨基酸序列如SEQ ID NO:29所示，所述TadA-ABE8e的核苷酸序列如SEQ ID NO:30所示。
所述MP1的核苷酸序列如SEQ ID NO:55所示；所述MP2的核苷酸序列如SEQ ID NO:56所示；所述MP3的核苷酸序列如SEQ ID NO:57所示；所述MP4的核苷酸序列如SEQ ID NO:58所示；所述MP5的核苷酸序列如SEQ ID NO:59所示。
结果表明，MP2的细胞存活率仅仅只有35.7％，突变率μ bp为3.6×10 -7，相对于MP1提高了3倍；MP3的细胞存活率为2.4％，突变率μ bp达到了2.76×10 -6，相对于MP1提高了23倍；MP4的细胞存活率为3％，突变率μ bp为1.8×10 -6，相对于MP1提高了15倍；MP5的细胞存活率仅仅只有1.7％，突变率μ bp达到了3.84×10 -6，相对于MP1提高了32倍。总体而言，在Cgl1289M和UGI的协同作用下，pmCDA1和TadA-ABE8e可显著提高C.glutamicum的突变率(见图7)。
1.3突变质粒突变谱测定
利用突变后利福平抗性平板上筛选得到的耐受利福平抗性的突变菌株的利福平抗性编码基因rpoB进行高通量测序，分析突变谱。
结果显示，发现MP1的突变谱较窄，主要的碱基突变类型集中在T:A-A:T和T:A-C:G。相比之下，MP3的突变碱基分布更均匀，覆盖了更多类型的突变，G:C-A:T的突变显著增加，这是因为DNA糖基化酶抑制剂UGI的表达，扰乱了碱基切除修复系统，同时胞嘧啶脱氨酶pmCDA1的表达增加了G:C-A:T的突变。同样，MP4由于表达了腺嘌呤脱氨酶TadA-ABE8e，也具备了比MP1更均匀的碱基突变分布，T:A-C:G和A:T-G:C的突变明显增加。MP5同时表达了胞嘧啶脱氨酶pmCDA1和腺嘌呤脱氨酶TadA-ABE8e，被赋予了更广泛的碱基突变分布，相对于MP1的碱基突变类型，G:C-A:T、T:A-C:G和A:T-G:C的碱基突变类型明显增加(见图8)。
同时，基于测序发现的rpoB突变位点基本囊括在已经报道了的能够获得利福平抗性的突变位点，其中也发现了三个新突变位点，如g1241a、c1286g和c1313t也能够使得C.glutamicum获得利福平抗性(表1)。这些结果表明，本发明开发的MPs突变质粒不仅拥有高的突变率，同时也具备广泛的突变谱。
表1利福平耐受突变株rpoB基因突变类型
Figure PCTCN2022102703-appb-000001
Figure PCTCN2022102703-appb-000002
注：下划线标注的突变位点表示的是无义突变，加粗字体标注的突变位点表示不包含在已经报道了rpoB突变文库中，其与突变位点均为已经报道的能够获得Rif r抗性的突变。
1.4 MP5突变质粒启动子优化
前期研究过程中，曾尝试利用弱诱导型启动子P arac来表达DNA聚合酶Cgl1289M，但在P arac调控下，并不能发挥突变能力。由于C.glutamicum中蛋白表达量较低，因此直接选择强启动子P tac调控DNA聚合酶Cgl1289M的表达，成功使得突变质粒MP1发挥了突变功能。当DNA聚合酶Cgl1289M和DNA糖基化酶抑制剂UGI处于表达过程中，才会发挥突变能力。因此，这两个酶的表达必须受到严格调控，且在C.glutamicum中能够使用的强诱导型且受到严格调控的启动子稀少，因此，并未对这两个酶的启动子进行优化。
为了找到合适的启动子来驱动pmCDA1和Tada-ABE8e的表达，选择了强诱导型启动子(P tac)、强组成型启动子(P tuf)、中等强度组成型启动子(P sod)和弱组成型启动子启动子(P zwf)来驱动胞嘧啶脱氨酶pmCDA1和腺嘌呤脱氨酶TadA-ABE8e的表达。
以质粒MP5为模板，以P5F/P5R为引物，通过反向PCR技术线性化缺失启动子P sod基因片段的重组质粒MP5，而后以C.glutamicum 13032基因组为模板分别扩增出启动子P tuf和P zwf基因片段，而后将启动子P tuf和P zwf基因片段和线性化MP5质粒进行同源重组连接，并转化E.coli BL21感受态细胞，分别构建重组质粒MPtuf和MPzwf。重组质粒MPtac以及MPdtac的构建直接以重组质粒MP5为模板，设计相 应的引物反向PCR扩增得到相应的线性化片段，而后利用同源重组连接试剂盒将所得到的线性化片段在37℃条件下孵育30min使其自连，而后化转到E.coli BL21感受态细胞，分别构建重组质粒MPtac和MPdtac。其中，P5F/P5R的引物序列具体如下：
P5-F：ttataacattttaattttattttctccattactgtct(SEQ ID NO:31)
P5-R：aaaggaggacaactaatgaccgac(SEQ ID NO:32)
通过将构建成功的重组质粒电转到C.glutamicum中并测定突变率发现，无论是强启动子P tac、P tuf，还是弱启动子P zwf，其突变率相对于MP5都没有明显提高。也就是说，适度强度的pmCDA1和Tada-ABE8e表达将有利于提高谷氨酸棒杆菌的突变率(见图9)。
1.5 RBS序列优化提高MP5突变率
利用在线网站RBS Calculator( https://www.denovodna.com/software/predict_rbs_calculator)针对DNA聚合酶Cgl1289M设计不同翻译起始率的RBS序列(表2)，以替换重组质粒MP5上Cgl1289M原始的RBS序列，所用反向PCR引物见表3，以MP5质粒为模板，利用相应的反向PCR引物进行PCR扩增，获得线性化片段并自连，分别构建出重组质粒MP5-RBS1、MP5-RBS2、MP5-RBS3、MP5-RBS4、MP5-RBS5和MP5-RBS6。测序验证正确后，将重组质粒分别电转至C.glutamicum感受态细胞，构建重组菌Cg-MP5-RBS1、Cg-MP5-RBS2、Cg-MP5-RBS3、Cg-MP5-RBS4、Cg-MP5-RBS5和Cg-MP5-RBS6。
表2不同翻译起始速率的RBS序列
Figure PCTCN2022102703-appb-000003
表3不同RBS序列所用反向PCR引物
Figure PCTCN2022102703-appb-000004
Figure PCTCN2022102703-appb-000005
表4不同强度RBS条件下的突变率和细胞存活率
Figure PCTCN2022102703-appb-000006
测定突变率发现，突变质粒MP5-RBS6的突变率最高，达到5.76×10 -6，相对于MP5提高了1.5倍(表4)。
1.5 MP5-RBS6突变质粒诱导条件优化
MPs突变质粒需要添加诱导剂IPTG来诱导DNA聚合酶Cgl1289M和DNA糖基化酶抑制剂UGI的表达，因而MPs的突变率会受到诱导剂的浓度和诱导温度的影响。因此，考察了不同浓度的IPTG添加量和诱导温度对于MP5-RBS6突变质粒突变率的影响。
MP5-RBS6突变质粒诱导方法：将含MP5-RBS6突变质粒的C.glutamicum在含氯霉素抗性的平板上划线活化，30℃培养箱倒置培养24～48h，挑取单菌落接种添加氯霉素的10mL BHI液体培养基，30℃、180rpm培养24h，按1％接种量转接添加氯霉素抗性的50mL BHI液体培养基，30℃、180rpm继续培养3h后添加不同量IPTG，放置不同温度下诱导12h。
考察不同诱导温度(16、20、25、30、35℃)对诱变效率的影响时，IPTG的添加浓度均控制在0.5mM。
结果表明，当诱导温度最适温度为30℃，诱变效率最高，也就是C.glutamicum最适生长的温度(图10中的A和C)。可能是因为无论是高温或者是低温都不利于C.glutamicum生长，同样也会阻碍DNA的正常复制，所以会导致突变率降低。
考察IPTG添加浓度(0.1、0.5、1.0、1.5、2.0mM)对诱变效率的影响时，诱导温度均控制在30℃。
结果显示，当IPTG的添加浓度为1mM时，诱变效率最高，突变率μ bp达到了6.12×10 -6(图10中的B和D)。
综上，当诱导剂添加浓度为1mM、诱导温度30℃时，MP5-RBS6质粒的诱变效率达到最高，突变率μ bp达到了6.12×10 -6，相对于野生型谷氨酸棒杆菌提高了153000倍。
1.7突变质粒消除体系构建
利用CRISPR体系中的温度敏感型复制起始位点RepA101替换MP5-RBS6自身的原始复制起始位点，构建重组菌Cg-MP5T。具体构建流程如下：
以质粒pCas9为模板，以repA101-F/repA101-R为引物PCR扩增出RepA 101基因片段，胶回收纯化获得的RepA 101基因片段与反向PCR扩增出的缺失原始质粒复制起始位点的线性化MP5片段进校同源重组连接，连接产物转化至E.coli BL21感受态细胞，构建重组质粒MP5T。测序验证正确后，将重组质粒MP5T电转至C.glutamicum感受态细胞，构建重组菌Cg-MP5T。其中，
repA101-F：tcagatccttccgtatttagccag(SEQ ID NO:51)
repA101-R：atgtctgaattagttgttttcaaagcaaat(SEQ ID NO:52)
所述温度敏感型复制起始位点RepA101的核苷酸序列如SEQ ID NO:53所示；所述MP5T的核苷酸序列如SEQ ID NO:54所示。
重组菌Cg-MP5T在BHI固体平板上划线活化，挑取单菌落转接到含有氯霉素抗性的BHI培养基中，30℃，180rpm培养18～24h。而后转接到无抗BHI培养基中，于42℃、180rpm条件下继续培养12h后，在无抗BHI平板上划线，30℃恒温培养箱中继续培养24～36h。最后，将生长在无抗BHI平板上的单菌落分别一一对应点在无抗BHI培养平板和含有氯霉素BHI培养平板上，30℃培养箱继续培养18～24h。其中，对应编号中氯霉素平板上不生长，无抗平板上生长的菌株即为MP5T质粒成功丢失的菌株。
实验表明，MP5T在30℃条件下可正常复制，42℃条件下则很容易就能够实现质粒消除(见图11)。也就是说，MP5T的成功构建使得MP5突变质粒不仅具备了高效的突变率以及广阔的突变谱，同时也具备了完成突变株筛选后，实现质粒的消除，不会使得突变质粒继续留存在细胞内持续造成本底突变，也为后续菌株的代谢工程改造以及遗传改造奠定了基础。
1.8 MP5T突变质粒与常规突变方法的比较
传统的诱变方式主要包括物理和化学诱变方法，为了更直观的比较MP5T与常规诱变方法的突变能力，进一步分析了EMS、MNNG、UV和ARTP四种诱变方法在C.glutamicum中的突变效果。
(1)甲基磺酸乙酯(EMS)诱变：
取1mL生长至对数中期C.glutamicum细胞离心并弃除上清液，并用1mL 100mM Tris-HCl(pH 7.0)缓冲液在冰上洗涤2次，然后添加100μL EMS并用移液枪轻轻吹吸混合均匀，并在200rpm，30℃条件下处理45min。而后离心并利用1mL 100mM Tris-HCl(pH 7.0)缓冲液在冰上洗涤2次以去除EMS残留，并将细胞浓度稀释10倍。而后接种于新鲜灭菌且不添加抗生素的BHI培养基中于180rpm，30℃条件下培养12-16h。
(2)1-甲基-3-硝基-1-亚硝基胍(MNNG)诱变：
取1mL生长至对数中期C.glutamicum细胞离心并弃除上清液，并用1mL 200 mM乙酸钠(pH 5.5)在冰上洗涤2次，然后添加至终浓度为1.6μg/mL MNNG并用移液枪轻轻吹吸混合均匀，并在30℃水浴条件下处理30min。而后收集细胞离心弃除MNNG上清，并利用1mL 100mM磷酸钾缓冲液(pH 7.0)在冰上洗涤2次以去除MNNG残留，并将细胞浓度稀释10倍。而后接种于新鲜灭菌且不添加抗生素的BHI培养基中于180rpm，30℃条件下培养12～16h。
(3)紫外(UV)诱变：
取1mL生长至对数中期C.glutamicum细胞离心并弃除上清液，并用1mL 100mM磷酸钾缓冲液(pH 7.0)在冰上洗涤2次，并利用1mL 100mM MgSO 4溶液重悬细胞。而后将细胞放置于灭菌的空培养皿中并在紫外线条件下处理100s(紫外灯距离培养皿高度为10cm)。处理结束后立刻转移细胞至新鲜灭菌且不添加抗生素的BHI培养基中于180rpm，30℃条件下培养12～16h，培养过程中要用锡箔纸包裹，防止见光发生回复突变。
(4)ARTP诱变：
取1mL生长至对数中期C.glutamicum细胞离心并弃除上清液，并用1mL无菌生理盐水在冰上洗涤2次。然后用移液枪吸取10μL细胞悬液并注入到灭菌的小铁片表面，并在ARTP作用30s。然后取出带有细胞悬液的小铁片并转移至新鲜灭菌且不添加抗生素的BHI培养基中于180rpm，30℃条件下恢复培养2～3h。
(5)MP5T诱变：
将含有MP5T质粒的C.glutamicum 13032细胞生长至对数中期，添加0.5mM IPTG诱导MP5T质粒表达，继续在30℃，180rpm条件下培养24～36h。
上述诱变方法所得到的细胞，经过细胞培养之后分别经过10倍梯度稀释涂布于无抗BHI固体平板以及含有终浓度10μg/mL利福平抗生素抗性平板上，倒置于30℃恒温培养箱静置培养24～36h之后，计算每个利福平抗性BHI平板和无抗BHI平板上生长的菌落数，并计算突变率。
结果表明，MP5T比其他四种诱变方法具有更高的突变率，突变频率f高达1.66×10 -3，也就是说，1000个突变子中就能得到1.66个阳性突变子。化学诱变剂EMS和MNNG在C.glutamicum中的突变频率不是很高，也有相关文献报道其突变谱比较窄，突变类型并不丰富(见图12)。而且，化学诱变剂如EMS和MNNG都是强致癌物，在环境中难以降解，对环境和人体具有持续的毒性作用，有着一定的潜在危险。虽然ARTP诱变具有相对广泛的突变谱(见图12)，但其突变率与化学诱变剂EMS和MNNG的差异不大，在C.glutamicum的突变率仍然较低。在利用紫外诱变筛选利福平耐受突变株的过程中，我们基本上没有得到阳性突变株，这可能是由于紫外诱变效率较低且突变方向具有序列偏好性，主要集中在C:G-A:T的突变。虽然与化学诱变剂相比，ARTP和UV诱变相对安全可靠，但UV和ARTP诱变通常构成单轮诱变，不能像化学诱变剂一样可以实现连续诱变。相比之下，MP5T突变质粒不仅突变率高、突变谱广、操作方法简单，而且可以可以使持续在细胞内实现突变的积累而不对操作者造成任何伤害，极大的缩短突变株文库构建时间，避免了资源和能源的浪费，大大提高了工作效率。
1.9筛选高产L-谷氨酸底盘细胞
基于实验室前期研究，野生型C.glutaminum 13032在初始发酵pH中性时，且发 酵过程中不调节pH，当细胞大量合成L-谷氨酸时，细胞微环境中的pH会持续降低，当pH为5.5时，会严重抑制细胞的生长。酸胁迫通常会引起细胞的一系列生理损伤，破坏生物大分子的结构，影响主要代谢途径中酶的活性，导致细胞内物质/能量代谢紊乱。因此，筛选能够耐受更低pH环境的突变菌株，提高细胞的耐酸性，对于C.glutaminum发酵生产L-谷氨酸有着重要的意义。
利用MP5T质粒连续诱变处理野生型C.glutaminum 13032 36h后，稀释涂布到pH 5.3 BHI固体平板上培养3天，而后在平板上挑取了47个单克隆于装有pH 5.3的液体BHI的48深孔板中继续培养24h，然后通过测定细胞密度OD 600。
结果表明，突变株NS-A1、NS-B4和NS-D1在pH 5.3环境下的生长状况最佳，生长强度明显优于出发菌株(图13中A)。
突变株NS-A1、NS-B4和NS-D1在30℃，180rpm培养12h后，用无菌生理盐水洗涤细胞两次，重悬于带有100mM homo-PIPES的BHI(pH 4.0)的培养基中使其初始细胞密度OD 600为0.2，并于30℃，180rpm培养，并分别在培养0h、2h、4h、6h和8h时取样。每个时间所取的细胞样品通过10倍梯度稀释并点板于BHI固体平板上，于30℃恒温培养箱中培养24h。培养结束后取出平板，并计算菌落数以及细胞存活率。细胞存活率＝不同时间酸处理细胞数/未进行酸处理的细胞数。
结果表明，NS-A1在pH 4的条件下胁迫8h，细胞存活率仍然高达43.75％，出发菌株C.glutaminum 13032的细胞存活率仅仅只有0.71％(图13中B)。
之后对NS-A1菌株进行5L发酵罐发酵，具体操作如下：取NS-A1保藏菌株冻管，在BHI固体培养皿上划线，分离单菌落。然后在BHI液体培养基中用接种环挑取单菌落并接入其中，于30℃，180rpm条件下培养12h，活化细胞。而后在10mL发酵种子培养基中接入活化后的细胞(接种量1％)，并于30℃，180rpm条件下培养12h，然后转移至200mL种子培养基中继续培养16-18h。最后在装有1.8L发酵培养基的5L发酵罐中将培养完成的200mL发酵种子液接全部接入。发酵罐发酵初始条件：30℃，搅拌速率600r/min，通气量1vvm，pH 7.3。当发酵培养基中葡萄糖浓度至20g/L时，发酵过程中的葡萄糖浓度控制在20～30g/L(流加补料培养基控制)。
结果显示，耐酸突变株NS-A1发酵48h能够积累L-谷氨酸35.3g/L，相对于野生型C.glutaminum 13032提高了16.7％(见图14)。
因此，我们成功利用MP5T，筛选得到了3株能够在pH 5.3条件下正常生长的突变菌株，且在一定程度上提高了L-谷氨酸的发酵水平，相对于正常的实验室进化筛选，缩短了菌种选育的时间，同时也为后续系统代谢工程改造打造L-谷氨酸高产菌株提供了耐酸型底盘细胞。这一谷氨酸棒杆菌高效诱变体系也可应用于其他氨基酸生产中。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (11)

  1. 一种谷氨酸棒杆菌高效突变体，其特征在于，所述突变体包括MP1～MP5和MP5T中的任意一种：
    所述MP1包括DNA聚合酶Cgl1289M，所述DNA聚合酶Cgl1289M的氨基酸序列如SEQ ID NO:13所示；
    所述MP2包括如下元件：所述Cgl1289M和尿嘧啶DNA糖基化酶抑制剂UGI；所述Cgl1289M的下游串联UGI，所述UGI的氨基酸序列如SEQ ID NO:25所示；
    所述MP3包括如下元件：所述Cgl1289M、UGI和胞嘧啶脱氨酶pmCDA1；所述Cgl1289M、UGI和pmCDA1依次串联；所述pmCDA1的氨基酸序列如SEQ ID NO:27所示；
    所述MP4包括如下元件：所述Cgl1289M、UGI和腺嘌呤脱氨酶TadA-ABE8e；所述Cgl1289M、UGI和TadA-ABE8e依次串联，所述TadA-ABE8e的氨基酸序列如SEQ ID NO:29所示。
    所述MP5包括如下元件：所述Cgl1289M、UGI、pmCDA1和TadA-ABE8e；所述Cgl1289M、UGI、pmCDA1和TadA-ABE8e依次串联；
    所述MP5T包括如下元件：温度敏感型复制起始位点RepA101、所述Cgl1289M、UGI、pmCDA1和TadA-ABE8e；所述RepA101、Cgl1289M、UGI、pmCDA1和TadA-ABE8e依次串联，所述RepA101的核苷酸序列如SEQ ID NO:53所示；
    优选的，所述DNA聚合酶Cgl1289M中RBS的核苷酸序列如SEQ ID NO:38所示；
    优选的，所述Cgl1289M和UGI共用一个启动子Ptac；所述pmCDA1和TadA-ABE8e共用一个启动子Psod；
  2. 编码权利要求1所述突变体的核苷酸，其特征在于，MP1～MP5和MP5T的核苷酸序列如SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59和SEQ ID NO:54所示。
  3. 含有权利要求1所述的突变体或权利要求2所述核苷酸的表达载体，其特征在于，所述表达载体包括pXMJ19、pDXW10、pEC-XK99E中的任意一种。
  4. 一种重组菌，其特征在于，所述重组菌表达权利要求1所述的突变体、权利要求2所述核苷酸或权利要求3所述表达载体；
    优选的，所述重组菌的出发菌株为谷氨酸棒杆菌13032。
  5. 一种谷氨酸棒杆菌高效突变体系，其特征在于，所述突变体系包括权利要求1所述突变体和诱变剂。
  6. 一种提高权利要求1所述突变体诱变效率的方法，其特征在于，包括如下步骤：所述突变体在诱导剂浓度为0.5～1.5mM，诱导温度为25～35℃下诱导。
  7. 权利要求4所述重组菌的构建方法，其特征在于，包括如下步骤：扩增突变体的核苷酸序列，连接到表达载体上，然后将表达载体转化到谷氨酸棒杆菌中，得到重组菌。
  8. 一种权利要求1所述MP5T在制备质粒消除体系中的应用；
    优选的，所述质粒消除体系还包括诱变剂；
    优选的，所述MP5T在培养温度为25～35℃条件下实现质粒的正常复制，当培养 温度为40～45℃时，质粒无法正常复制，从而实现突变质粒的消除。
  9. 权利要求1所述突变体、权利要求2所述核苷酸、权利要求3所述表达载体或权利要求5所述突变体系在筛选高产谷氨酸底盘细胞中的应用。
  10. 权利要求1所述突变体、权利要求2所述核苷酸、权利要求3所述表达载体或权利要求5所述突变体系在生产氨基酸中的应用。
  11. 权利要求1所述突变体、权利要求2所述核苷酸、权利要求3所述表达载体或权利要求5所述突变体系在筛选耐酸型菌株中的应用；
    优选的，所述耐酸性菌株为高产谷氨酸耐酸性菌株。
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