Corynebacterium glutamicum whole genome random mutation tool and application thereof

Abstract

The invention discloses a corynebacterium glutamicum whole genome random mutation tool and application thereof, and belongs to the technical field of genetic engineering and biological mutagenesis. DsDNA is unwound in the transcription process based on DNA helicase, DNA adenine deaminase and the DNA helicase are fused, and whole-genome random mutation of corynebacterium glutamicum is achieved. The whole genome random mutation method disclosed by the invention is applied to mutagenesis of an original strain of Corynebacterium glutamicum ATCC 13032, and a mutant strain of which the biomass is 1.95 times of that of WT (OD600 is 2.2) and 1.99 times of that of WT / pXMJ19 (OD600 is 2.16) when being cultured under the condition that the pH value is 5.5 is obtained. The corynebacterium glutamicum whole genome random mutation method disclosed by the invention has important guiding significance for screening other mutation strains with enhanced robustness.

Description

Technical Field

[0001] The present invention relates to a Corynebacterium glutamicum whole genome random mutation tool and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] In nature, evolution is the ultimate designer and creator of organisms, and mutation is the fundamental source of all variation driving evolution. Random genomic mutations confer genetic diversity to cells, providing ideal phenotypes for both basic research and industrial production. However, genomic stability typically results in low mutation frequencies in cells, particularly in prokaryotes, whose replication, transcription, and translation processes exhibit higher fidelity than eukaryotic cells. This makes it difficult for researchers to rapidly generate phenotypically diverse organisms through spontaneous mutation. In recent years, the development of various continuous mutagenesis techniques has greatly improved the efficiency of random genomic mutagenesis, accelerating microbial evolution in areas such as expanding substrate utilization, improving stress resistance, and enhancing product synthesis. These techniques have also played a significant role in exploring the relationship between gene and phenotype. C. glutamicum is an important industrial microorganism, particularly in the amino acid industry. Traditional C. glutamicum genome evolution engineering relies on physical mutagenesis, chemical mutagenesis, heavy ion irradiation, ARTP mutagenesis, and genome shuffling. However, these methods are not continuous, significantly limiting the acquisition of superior mutations. ALE is a widely used method for continuous evolution of the C. glutamicum genome, but it relies on spontaneous mutations in the genome, making it difficult for researchers to quickly obtain ideal phenotypes on a laboratory timescale.

[0003] Starting from the tRNA adenine deaminase from E. coli, the researchers obtained a highly active DNA adenine deaminase TadA-8e through phage-assisted continuous and discontinuous evolution. It deaminates adenine (A) using DNA as a substrate. DNA adenine deaminase deaminates A to produce hypoxanthine (I). I cannot be effectively removed by any known DNA glycosylase. During the subsequent DNA replication and repair process, I will be recognized as guanine (G) for reading and replication, and then pair with the corresponding cytosine (C), thereby realizing the conversion of A:T base pairs to G:C base pairs.

[0004] The expression of TadA-8e alone has a poor mutagenic effect on the genome, which is attributed to the fact that TadA-8e is a single-stranded DNA (ssDNA) deaminase. In most cases, DNA exists in a double helix form, and only during transcription, replication and recombination is the double helix briefly unwound to expose ssDNA. Summary of the Invention

[0005] Technical problem: The technical problem to be solved and the goal to be achieved by the present invention.

[0006] The present invention provides a whole-genome random mutation method for Corynebacterium glutamicum. The method is used to perform genome mutagenesis on the original strain of C. glutamicum ATCC 13032 to obtain a mutant strain whose biomass is 1.91 times that of the original strain when grown under pH 5.5.

[0007] Technical solution: The complete technical means and methods of the present invention.

[0008] The present invention provides a tool for random mutation of the Corynebacterium glutamicum genome, which comprises a plasmid vector skeleton on which are assembled and connected: a nucleotide sequence encoding DNA adenine deaminase and a nucleotide sequence encoding Corynebacterium glutamicum DNA helicase.

[0009] In one embodiment of the present invention, the DNA adenine deaminase comprises any one of TadA8e, TadA7.10, TadA8.17, TadA8.20, and TadA9;

[0010] The DNA helicase includes any one of Cgl0025, Cgl0141, Cgl0776, Cgl0779, Cgl0846, Cgl0854, Cgl0894, Cgl1156, Cgl1326, Cgl1927, and Cgl2519;

[0011] In one embodiment of the present invention, the DNA adenine deaminase is TadA8e; the amino acid sequence of the DNA adenine deaminase TadA8e is shown in SEQ ID NO.1;

[0012] MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSSGGSSGSETPG TSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN

[0013] In one embodiment of the present invention, the DNA helicase is Cgl2519; the amino acid sequence of the DNA helicase Cgl2519 is shown in SEQ ID NO.2;

[0014] MSTATIALQRQLVNRDLPRLVDALEPLMERRPTFAIMKGRSNYLCMNKVARQEELNQEDALIEQEDISWLGKHIVRLNEWANETETGDRDDLDPGVPDLAWKQVSVTARECIGASRCPHGEDCFAEIARGKAKEADVVVTNHALL AIDALSDVSVLPEHDVVVIDEAHELDGRITAVASAEITVNSLNLAARRASKLDSDKREERVQEIAGDLETLLQTMQPGRWNDMDEGSKGTLVALKDALWALRAQIAGAPEGEAANDPERFAERQNLSNHLMEIHDANVRILEVFA EEDPSKQYDVVWHNHDDRRGDSLNVAPLSVAGLLHEKLFAENTVVLASATLTIGGNFNAMAASWGLPKGSWDSMDAGTPFDPAKSGILYTARHLPDPGRDGLPEETLDEIYELITAAGGRTLGLFSSKRAAEQATKAMRLRLPFD VLCQGDDNTAALVKKFSDSENTCLFGTLTLWQGVDVPGRSLSLVLIDRIPFPRPDDPLLQARKEAADAEGRNGFMEVAATHAALLMAQGAGRLLLRHVGDRGVVAVLDHRLSTKRYGGFLRFSMPRFWETTNPETVRAALKRLVTK

[0015] In one embodiment of the present invention, DNA adenine deaminase TadA8e is fused to the C-terminal endometrium of DNA helicase Cgl2519 via a flexible linker.

[0016] In one embodiment of the present invention, the amino acid sequence of the linker is shown in SEQ ID NO.3:

[0017] PKKKRKVGGGGSGGGGSAEYVRALFDFNGNDEEDLPFKKGDILRIRDKPEEQWWNAEDSEGKRGM IPVPYVEKYSGDYKDHDGDYKDHDIDYKDDDDKSR

[0018] In one embodiment of the present invention, the plasmid vector is selected from any one of pXMJ19, pDXW10 and pEC-XK99E.

[0019] The present invention also provides a recombinant Corynebacterium glutamicum, which is obtained by transforming the above-mentioned Corynebacterium glutamicum genome random mutation tool into Corynebacterium glutamicum.

[0020] The present invention also provides a method for improving the acid resistance, high temperature resistance, oxidation resistance and / or osmotic pressure resistance of Corynebacterium glutamicum, wherein the method comprises transforming Corynebacterium glutamicum with the above-mentioned Corynebacterium glutamicum genome random mutation tool.

[0021] The present invention also provides the use of the above-mentioned random mutation tool for the genome of Corynebacterium glutamicum in improving the robustness and / or biosynthesis efficiency of Corynebacterium glutamicum.

[0022] The present invention also provides the use of the above-mentioned random mutation tool for the genome of Corynebacterium glutamicum in screening strains with high robustness and / or biosynthetic efficiency.

[0023] The present invention also provides a method for screening Corynebacterium glutamicum with high acid resistance, which comprises transforming Corynebacterium glutamicum with the above-mentioned Corynebacterium glutamicum genome random mutation tool to obtain recombinant Corynebacterium glutamicum; and continuously subculturing the obtained recombinant Corynebacterium glutamicum under acidic conditions.

[0024] The present invention also provides a method for improving the rifampicin-resistant mutation rate of Corynebacterium glutamicum, characterized in that the method comprises transforming Corynebacterium glutamicum with the above-mentioned Corynebacterium glutamicum genome random mutation tool.

[0025] In one embodiment of the present invention, the method comprises fusing DNA adenine deaminase TadA8e and the C-terminal endometrium of DNA helicase Cgl2519 via a flexible linker to obtain a fusion protein;

[0026] After the fusion protein is connected to the pXMJ19 plasmid, a recombinant vector is obtained; and the obtained recombinant vector is introduced into Corynebacterium glutamicum.

[0027] Beneficial effects

[0028] The present invention provides a method for random mutation of the entire genome of Corynebacterium glutamicum, which significantly increases the genome mutation rate. Using this method, mutants with improved robustness can be obtained in a short period of time through continuous evolution. The continuous evolution method for improving the robustness of Corynebacterium glutamicum is applied to evolve acid-tolerant mutants. It does not require a complex experimental setup and, after only 312 hours (13 generations), mutants with a significant growth advantage in a culture medium containing pH 5.5 are obtained. Therefore, the method of the present invention reduces the time used to evolve acid-tolerant mutant strains and greatly improves the efficiency of screening acid-tolerant strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Screening for the optimal DNA helicase and the optimal TadA8e fusion orientation.

[0030] Figure 2 : Acid tolerance test of C. glutamicum ATCC 13032.

[0031] Figure 3 : The process of continuous evolution of acid-tolerant mutants using P19-Cgl2519-TadA8e.

[0032] Figure 4 : Comparison of acid tolerance between mutant strain NS-Cg and wild-type strain. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0035] The hosts used for plasmid cloning and expression in the present invention are Corynebacterium glutamicum ATCC13032 and Escherichia coli JM109.

[0036] The plasmid used for gene expression in the present invention is pXMJ19.

[0037] The culture medium involved in the following examples is as follows:

[0038] LB medium (g / L): 10% peptone, 10% NaCl, 5% yeast extract. For solid medium, add 1.5%-2% agar powder. Sterilize at 115°C for 20 minutes. For E. coli culture.

[0039] BHI medium (g / L): BHI powder 38.5. If solid medium is required, add 1.5%-2% agar powder. Sterilize at 115°C for 20 minutes. For the cultivation of C. glutamicum.

[0040] LBG medium (g / L): 10% peptone, 10% NaCl, 5% yeast extract, 5-10% glucose. For solid medium, add 1.5%-2% agar powder. Sterilize at 115°C for 20 minutes. Used for the cultivation and mutagenesis of C. glutamicum.

[0041] CGXII medium (g / L): MOPS 42%, NH4SO4 20%, glucose 5%, urea 5%, K2HPO4 1%, KH2PO4 1%, MgSO4·7H2O 0.25%, MnSO4·H2O 0.01%, FeSO4·7H2O 0.01%, CaCl2 0.01%, protocatechuic acid 0.03%, ZnSO4·7H2O 1%, biotin 0.2%, CuSO4 0.2%, NiCl2·6H2O 0.02%. For solid medium, add 1.5%–2% agar powder. Sterilize at 115°C for 20 min.

[0042] Epo medium (g / L): glycine 30, tryptone 10, NaCl 10, glucose 5, yeast extract 5, isoniazid 4, Tween-80 1 mL / L. Used for preparing competent cells.

[0043] Kanamycin (Kan), chloramphenicol (Chl), and rifampicin (Rif) were added to the culture medium at a final concentration of 50 μg / mL as needed. Target protein expression was induced by adding 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) to the culture medium.

[0044] The method for preparing E. coli competent cells according to the present invention is as follows:

[0045] To prepare competent E. coli cells, activate the cells by streaking onto LB plates from frozen tubes. After 12 hours of incubation, pick a single colony from the LB plate and inoculate it into LB liquid medium. Incubate at 37°C, 180 rpm, and 10 hours. Transfer a 1% inoculum to fresh 50 ml of LB liquid medium and incubate at 37°C, 180 rpm, for 1.5-2 hours until the OD reaches 0. 600= approximately 0.5. Place the bacterial solution on ice for 10 minutes, then centrifuge at 6000g for 10 minutes at 4°C to collect the cells. Discard the supernatant, add 5 mL of pre-chilled 0.05 mol / L CaCl2 solution, gently resuspend the cells, place on ice for 5-10 minutes, and then centrifuge at 6000g for 10 minutes at 4°C to collect the cells. Discard the supernatant, add 5 mL of pre-chilled 0.05 mol / L CaCl2 solution containing 15% glycerol, gently resuspend the cells, and aliquot into 80 μl tubes. Use immediately or store in a -80°C freezer.

[0046] The present invention relates to a method based on rpoB / Rif r Systematic testing of genome mutation rates

[0047] Screen rifampicin-resistant mutant strains to test the mutation rate. Activate the strain to be tested by streaking on a BHI plate and incubate at 30°C for 24-48 hours. Take a single colony of the strain to be tested and inoculate it into BHI liquid culture medium and culture it at 30°C overnight. Inoculate the bacterial solution into LBG medium at a 1% inoculum volume and continue to culture. After 3 hours, add IPTG to a final concentration of 0.5mM to induce the expression of the target protein. After 24 hours of induction, take the bacterial solution and perform gradient dilution with sterile saline, and take 100ul each and spread it on BHI non-antibiotic and BHI+8μg / mL rif plates. Invert the plate and culture at 30°C until a single colony grows. Count the single colonies on the plate and calculate the mutation rate.

[0048] Mutation rate calculation formula = (number of colonies on BHI+8μg / mL rif plate × dilution factor) / (number of colonies on BHI non-antibiotic plate × dilution factor)

[0049] The continuous evolution method of the acid-tolerant mutant strains of the present invention is as follows:

[0050] C. glutamicum containing the mutagenic plasmid P19-Cgl2519-TadA8e was used as the experimental group, and C. glutamicum containing the pXMJ19 empty plasmid was used as the control group. Three groups of continuous evolution experiments were performed on the experimental group and the control group respectively. The three groups of continuous evolution experiments represent three different mutation situations to increase the possibility of obtaining the desired mutants. Three colonies containing empty plasmid control bacteria and three experimental bacterial colonies containing P19-Cgl2519-TadA8e were picked from the BHI plate and inoculated into BHI liquid culture medium containing 10μg / mL Chl, and cultured at 200rpm and 30℃ for 18-24h. The cells were then transferred to CGXII culture medium at a 1% inoculation rate and cultured as seeds for 18-24h. The seeds were then transferred to CGXII culture medium, and 0.5mM IPTG, 10μg / mL Chl, pH 5.5 were added as evolutionary pressure. The OD600 The highest bacterial solution was used as seed to transfer to the next generation, and the initial OD 600 The pH value was 0.1. Evolution was terminated when the experimental strain showed a significant growth advantage over the control strain under low pH stress. The culture was appropriately diluted with sterile saline and plated on CGXII agar plates at pH 5.5. Culture was continued until single colonies emerged, and larger colonies were selected for subsequent tolerance testing.

[0051] Example 1: Screening for the optimal DNA helicase and optimal TadA8e fusion orientation

[0052] 1. Construction of fusion expression of DNA helicase and TadA8e

[0053] During replication, DNA helicase breaks hydrogen bonds between dsDNA, unwinding the DNA fragments to form transient ssDNA, which then slides along the DNA template. DNA helicase acts as an "anchor protein" for TadA8e, positioning TadA8e around ssDNA during replication to deaminize adenine and generate base transitions.

[0054] Eleven genes annotated as DNA helicases were selected from the Corynebacterium glutamicum genome (BA000036.3), including Cgl0025, Cgl0141, Cgl0776, Cgl0779, Cgl0846, Cgl0854, Cgl0894, Cgl1156, Cgl1326, Cgl1927, and Cgl2519 (SEQ ID NO. 2, gene sequence: SEQ ID NO. 5);

[0055] TadA8e (SEQ ID NO.1) was fused to the N-terminus or C-terminus of the above-mentioned DNA helicase via a flexible linker of 100 amino acid residues (SEQ ID NO.3), and 22 different fusion expression plasmids were constructed on the pXMJ19 plasmid.

[0056] (1) TadA8e (SEQ ID NO. 1) was fused to the N-terminal endometrium of DNA helicase via a 100 AA linker. Taking the P19-Cgl2519-TadA8e-N plasmid as an example, the specific steps are as follows:

[0057] 1) A 100-amino acid flexible linker was synthesized by GeneWeiZ to obtain a 100-AA fragment.

[0058] The TadA8e gene fragment (SEQ ID NO. 4) was amplified using the MP6 plasmid (purchased from Addgene) as a template using the primer pair TadA8e-F / TadA8e-R.

[0059] Using the pXMJ19 plasmid as a template, inverse PCR was performed with primer pair P19-F / P19-R to obtain a linearized plasmid fragment, which was then digested with DpnI for 30 min.

[0060] 2) The three fragments obtained in step 1) were ligated using the ClonExpress II One-Step Cloning Kit and chemically transformed into E. coli JM109. After sequencing verification, the recombinant plasmid, P19-100AA-TadA8e, was constructed. Using the P19-100AA-TadA8e plasmid as a template, inverse PCR was performed with primer pair P19-100AA-TadA8e-F / P19-100AA-TadA8e-R to linearize the plasmid fragment, which was then digested with DpnI for 30 minutes.

[0061] The gene fragment Cgl2519 was amplified using the C. glutamicum ATCC 13032 genome as a template using the primer pair Cgl2519-F / Cgl2519-R.

[0062] The two fragments were ligated using the ClonExpress II One-Step Cloning Kit and chemically transformed into E. coli JM109. After sequencing verification, a recombinant vector fused with TadA8e and the N-terminus of the DNA helicase was constructed: P19-Cgl2519-TadA8e-N. The primer sequences involved are:

[0063] P19-F:GGTTGTCCTCCTTTcatggtctgtttcctgtgtg

[0064] P19-R:tctagagtcgacctgcaggcatg

[0065] TadA8e-F:ATGTCTGAAGTGGAGTTTCCCCATG

[0066] TadA8e-R:TTAATTGATGGAGGACTGTGCCTTC

[0067] P19-100AA-TadA8e-F: CCAAAGAAGAAACGCAAGGTCGGTGGTGGTGGCTCTGGC

[0068] P19-100AA-TadA8e-R:GGTTGTCCTCCTTTgcatgcaagcttaattaattctgtttcctgtgtg

[0069] Cgl2519-N-F: cagaattaattaagcttgcatgcAAAGGAGGACAACCAtgtctactgcgacgatcgcgttg

[0070] Cgl2519-N-R: CGACCTTGCGTTTCTTCTTTGGcttagtcaccaatcgtttgagtgccgcgc

[0071] According to the above method, P19-Cgl0025-TadA8e-N, P19-Cgl0141-TadA8e-N, P19-Cgl0776-TadA8e-N, P19-Cgl0779-TadA8e-N, P19-Cgl0846-TadA8e-N, P19-Cgl0854-TadA8e-N, P19-Cgl0894-TadA8e-N, P19-Cgl1156-TadA8e-N, P19-Cgl1326-TadA8e-N, and P19-Cgl1927-TadA8e-N were respectively prepared. The primer sequences involved are as follows:

[0072] Cgl0025-N-F: gcaaccgagcgtacttgaaggctCCAAAGAAGAAACGCAAGGTC

[0073] Cgl0025-N-R: catgatggggcactggttccacGGTTGTCCTCCTTTgcatgcaagcttaattaattctg

[0074] Cgl0141-N-F: gcgcgcaccaagaatcgcatgCCAAAGAAGAAACGCAAGGTCGG

[0075] Cgl0141-N-R: gaaaattgccaattcttgaactcatGGTTGTCCTCCTTTgcatgcaagcttaattaattctg

[0076] Cgl0776-N-F: gaaatcctacgcatggatgacacGGTTGTCCTCCTTTgcatgcaagcttaattaattc

[0077] Cgl0776-N-R: gcccggctgctcagtcaggaaCCAAAGAAGAAACGCAAGGTCGG

[0078] Cgl0779-N-F:gctcgcgattttgggagcacttCCAAAGAAGAAACGCAAGGTCG

[0079] Cgl0779-N-R:cgtcgaggtcctgcaaattgatcacGGTTGTCCTCCTTTgcatgcaagcttaattaattc

[0080] Cgl0846-N-F:cgtggaggcttcggacggcgcCCAAAGAAGAAACGCAAGGTCGGTGGTG

[0081] Cgl0846-N-R:cggaatcgggaaagaatgcttttagccatGGTTGTCCTCCTTTgcatgcaagcttaattaattc

[0082] Cgl0854-N-F:ggcgtcccgatggagaagctaCCAAAGAAGAAACGCAAGGTCGGTG

[0083] Cgl0854-N-R:ggggtaaaaggagaagtattcatGGTTGTCCTCCTTTgcatgcaag

[0084] Cgl0894-N-F:gttcgctttagcgagtaggggaCCAAAGAAGAAACGCAAGGTCGG

[0085] Cgl0894-N-R:ggcttcttccctcgttgcgttcatGGTTGTCCTCCTTTgcatgcaagcttaattaattc

[0086] Cgl1156-N-F:ggcggttaccgtggcggacgcgacCCAAAGAAGAAACGCAAGGTCGGTGG

[0087] Cgl1156-N-R:cgttgacgttctcggtattactcatGGTTGTCCTCCTTTgcatgcaagcttaattaattctg

[0088] Cgl1326-N-F:gatcaaccaggaatacctggaaaagagcCCAAAGAAGAAACGCAAGGTCGGTGGTG

[0089] Cgl1326-NR:gcaaacgctcgtcatgccaacccaacatGGTTGTCCTCCTTTgcatgcaagcttaattaattc

[0090] Cgl1927-NF:ccagaaggctattgccaagcttcgaCCAAAGAAGAAACGCAAGGTCGGTGG

[0091] Cgl1927-NR:ggcagtttcagaagtagtcatGGTTGTCCTCCTTTgcatgcaagcttaattaattc

[0092] (2) TadA8e (SEQ ID NO.1) was fused to the C-terminal end of DNA helicase to form P19-Cgl2519-TadA8e-C

[0093] Taking plasmid construction as an example, the specific steps are as follows:

[0094] 1) A 100-amino acid flexible linker was synthesized by GeneWeiZ to obtain a 100-AA fragment.

[0095] The TadA8e gene fragment was amplified using the MP6 plasmid (purchased from Addgene) as a template using the primer pair TadA8e-F / TadA8e-R.

[0096] Using the pXMJ19 plasmid as a template, inverse PCR was performed with primer pair P19-F / P19-R to obtain a linearized plasmid fragment, which was then digested with DpnI for 30 min.

[0097] 2) The three fragments obtained in step 1) were ligated using the ClonExpress II One-Step Cloning Kit and chemically transformed into E. coli JM109 to construct P19-TadA8e-100AA. Reverse PCR was performed using the P19-TadA8e-100AA plasmid as a template with primer pair P19-TadA8e-100AA-F / P19-TadA8e-100AA-R to linearize the plasmid fragment, which was then digested with DpnI for 30 minutes.

[0098] The gene fragment Cgl2519 was amplified using the C. glutamicum ATCC 13032 genome as a template using the primer pair Cgl2519-F / Cgl2519-R.

[0099] The two fragments were ligated using the ClonExpress II One-Step Cloning Kit and chemically transformed into E. coli JM109 to construct a recombinant vector fused with the C-terminus of TadA8e and DNA helicase: P19-Cgl2519-TadA8e-C. The primer sequences involved are:

[0100] P19-F:GGTTGTCCTCCTTTcatggtctgtttcctgtgtg

[0101] P19-R:tctagagtcgacctgcaggcatg

[0102] TadA8e-F:gcttgcatgcAAAGGAGGACAACCATGTCTGAAGTGGAGTTCTCCC

[0103] TadA8e-R:CACCACCGACCTTGCGTTTCTTCTTTGGATTGATGGAGGACTGTGCCTTCT

[0104] P19-TadA8e-100AA-F: GCGGGATTTATCGTCGTCGTCCTTATAGTCGATG

[0105] P19-TadA8e-100AA-R:agcttggctgttttggcggatgagagaagattttcagcctg

[0106] Cgl2519-CF:catccgccaaaacagccaagctttacttagtcaccaatcgtttgag

[0107] Cgl2519-CR:CTATAAGGACGACGACGATAAATCCCGCgtgtctactgcgacgatcgcgttg

[0108] According to the above method, P19-Cgl0025-TadA8e-C, P19-Cgl0141-TadA8e-C, P19-Cgl0776-TadA8e-C, P19-Cgl0779-TadA8e-C, P19-Cgl0846-TadA8e-C, P19-Cgl0854-TadA8e-C, P19-Cgl0894-TadA8e-C, P19-Cgl1156-TadA8e-C, P19-Cgl1326-TadA8e-C, and P19-Cgl1927-TadA8e-C were prepared respectively. The primer sequences involved are as follows:

[0109] Cgl0025-C-F:GGACGACGACGATAAATCCCGCgtggaaccagtgccccatcatg

[0110] Cgl0025-C-R:cttctctcatccgccaaaacagccaagcttcaagccttcaagtacgctcgg

[0111] Cgl0141-C-F:GGACGACGACGATAAATCCCGCatgagttcaagaattggcaattttc

[0112] Cgl0141-C-R:cttctctcatccgccaaaacagccaagctctacatgcgattcttggtgcgcgcagtg

[0113] Cgl0776-C-F:CTATAAGGACGACGACGATAAATCCCGCgtgtcatccatgcgtaggatttctcctc

[0114] Cgl0776-C-R:ctctcatccgccaaaacagccaagcttcattcctgactgagcagccgggctag

[0115] Cgl0779-C-F:ctctcatccgccaaaacagccaagctctaaagtgctcccaaaatcgcgag

[0116] Cgl0779-C-R:GACTATAAGGACGACGACGATAAATCCCGCgtgatcaatttgcaggacctcg

[0117] Cgl0846-C-F:ctctcatccgccaaaacagccaagctttagcgccgtccgaagcctccacgaaaagt

[0118] Cgl0846-C-R:CTATAAGGACGACGACGATAAATCCCGCatggctaaaagcattctttcccgattccgac

[0119] Cgl0854-C-F:GGACGACGACGATAAATCCCGCatgaatacttctccttttaccccagg

[0120] Cgl0854-CR:gttcctggccttttgctggccttttgcctatagcttctccatcgggacgccacc

[0121] Cgl0894-CF:GGACGACGACGATAAATCCCGCatgaacgcaacgagggaagaagcc

[0122] Cgl0894-CR:ctggccttttgctggccttttgcctatcccctactcgctaaagcgaac

[0123] Cgl1156-CF:AGGACGACGACGATAAATCCCGCatgagtaataccgagaacgtcaacg

[0124] Cgl1156-CR:ctggccttttgctggccttttgcttagtcgcgtccgccacggtaaccgc

[0125] Cgl1326-CF:GGACGACGACGATAAATCCCGCatgttgggttggcatgacgagcg

[0126] Cgl1326-CR:cctggccttttgctggccttttgctcagctcttttccaggtattcctgg

[0127] Cgl1927-CF:CTATAAGGACGACGACGATAAATCCCGCatgactacttctgaaactgccccatc

[0128] Cgl1927-CR:ctctcatccgccaaaacagccaagctttatcgaagcttggcaatagccttctggattc

[0129] As a control, a recombinant vector expressing only TadA8e, P19-TadA8e, was prepared according to the above method.

[0130] The primer sequences involved are:

[0131] P19-F:GGTTGTCCTCCTTTgcatgcaagcttaattaattctgtttcctgtgtg

[0132] P19-R:gcaaaaggccagcaaaaggccaggaaccgtaaaaag

[0133] TadA8e-F:gaaacagaattaattaagcttgcatgcAAAGGAGGACAACCATGTCTGAAGTGGAGTTCTCCCATG

[0134] TadA8e-R: gttcctggccttttgctggccttttgcTTAATTGATGGAGGACTGTGCCTTCTTTTG

[0135] 2. Comparison of mutation rates of different DNA helicases fused to TadA8e at different orientations

[0136] The recombinant plasmids constructed above were electroporated into C. glutamicum ATCC 13032 to construct recombinant strains. The successfully verified recombinant strains and the wild-type strain (C. glutamicum ATCC 13032) were streaked onto BHI plates and activated, and cultured at 30°C for 24-48 hours.

[0137] Take a single colony of the strain to be tested and inoculate it into BHI liquid medium, culture it at 30℃ overnight (24h); obtain seed liquid. Inoculate the prepared seed liquid into LBG medium at an inoculum volume of 1% (v / v) and continue to culture (conditions are: 30℃, 180rpm). After 3 hours, add IPTG to a final concentration of 0.5mM to induce the expression of the target protein. After induction at 30℃ for 24h, take the bacterial solution and perform gradient dilution with sterile physiological saline, and take 100ul each and spread it on BHI without antibiotics and BHI+8μg / mL rif plates. Invert the plate and culture it at 30℃ until a single colony grows. Count the single colonies on the plate and calculate the mutation rate.

[0138] The results are shown in the following table:

[0139] Table 1: Mutation rates of strains containing different vectors

[0140]

[0141]

[0142] The results showed that after 24 h of IPTG induction, the expression of some fusion devices increased the rate of rifampicin-resistant mutations compared with the control strain expressing independent TadA8e ( Figure 1 ). Notably, the recombinant strain expressing P19-Cgl2519-TadA8e-C showed a significantly increased mutation rate of 6.53×10-6 , which is 21.20 times higher than when only TadA8e is expressed, and is 2.5 times higher than the spontaneous mutation rate of the wild-type strain (3.55×10 -8 ) is 183.90 times.

[0143] Therefore, P19-Cgl2519-TadA8e was subsequently selected to continuously evolve acid-tolerant mutants.

[0144] Example 2: Acid tolerance test of C. glutamicum ATCC 13032

[0145] Determine the acid tolerance of wild-type C.glutamicum ATCC 13032. First, activate C.glutamicum ATCC 13032 by streaking on a BHI plate and incubate at 30°C for 24-48 hours. Pick a single colony and inoculate it into BHI liquid medium. Incubate it at 30°C overnight (24 hours) to obtain a bacterial solution. Inoculate the bacterial solution with a 1% (v / v) inoculum into CGXII liquid medium at pH 6.0, 5.8, 5.6, 5.5, 5.4, 5.3 and 5.2. Incubate it at 30°C, 200 rpm for 24 hours and measure the OD value. 600 Each series of experiments consisted of three parallel experiments. The results showed that the growth of the strain was significantly inhibited as the pH gradually decreased. When the pH was 5.2, the cell growth rate decreased by 95.85%, and the OD600 was only 0.28 ( Figure 2 ).

[0146] Acid tolerance test of recombinant strain: The method is the same as that of wild type. The results show that the acid tolerance of recombinant strain before evolution is not significantly different from that of wild type strain ( Figure 2 ).

[0147] Example 3: Continuous evolution of acid-tolerant mutants using P19-Cgl2519-TadA8e

[0148] Continuous evolutionary process Figure 3 As shown, C. glutamicum ATCC 13032 containing the mutagenic plasmid P19-Cgl2519-TadA8e-C was used as the experimental group (E group, C. glutamicum ATCC 13032 / P19-Cgl2519-TadA8e-C); C. glutamicum ATCC 13032 containing the empty plasmid pXMJ19 was used as the control group (NC group, C. glutamicum ATCC 13032 / pXMJ19).

[0149] Three sets of continuous evolution experiments were performed simultaneously on the experimental group and the control group. The three sets of continuous evolution experiments represented three different mutation situations to increase the possibility of obtaining the desired mutants. The specific steps are as follows:

[0150] Three control colonies containing an empty plasmid and three experimental colonies containing P19-Cgl2519-TadA8e-C were picked from a BHI plate and inoculated into BHI liquid medium containing 10 μg / mL Chl. The cells were cultured at 200 rpm and 30°C for 24 hours. The cells were then transferred to CGXII medium at a 1% (v / v) inoculum as seeds and cultured for another 24 hours to prepare a seed solution. The seed solution was then transferred to CGXII medium (pH 5.5) at a 1% (v / v) inoculum and cultured at 30°C for 24 hours with 0.5 mM IPTG and 10 μg / mL Chl. The strains were continuously acclimated using pH 5.5 as the evolutionary pressure to prepare a bacterial solution.

[0151] The OD values ​​of the three experiments in each group were 600 The highest bacterial solution was used as the seed solution to transfer to the next generation (transferred to CGXII medium with pH 5.5), and the initial transfer OD 600 was 0.1, and the culture was continued for 312 h (13 generations) according to the above conditions.

[0152] The results show:

[0153] After continuous evolution for 312 h (13 generations) in a pH 5.5 culture medium, the recombinant bacteria (C. glutamicum ATCC 13032 / P19-Cgl2519-TadA8e-C) expressing P19-Cgl2519-TadA8e-C finally showed a significant growth advantage.

[0154] And by Figure 3 It can be seen that within the same period of time, the OD600 value of the control strain, that is, the recombinant strain carrying the empty plasmid, did not change, indicating that the acid resistance of the wild-type strain was not improved during the same period of continuous evolution.

[0155] The culture was diluted appropriately with sterile saline and plated on a CGXII agar plate at pH 5.5. Culture was continued until a single colony grew, and the largest single colony (NS-Cg) was selected for subsequent tolerance testing.

[0156] Example 4: Testing and comparing the acid tolerance differences between the acid-tolerant mutant strain NS-Cg and the wild-type strain

[0157] 1. Single colonies of the evolved NS-Cg and C. glutamicum ATCC 13032 original bacteria (WT) from Example 3 and the control strain C. glutamicum ATCC 13032 / pXMJ19 (WT / pXMJ19) carrying an empty plasmid were picked and inoculated into BHI liquid medium. After culturing at 30°C and 200 rpm for 24 h, the bacterial solution was inoculated at a 1% (v / v) inoculum into CGXII liquid medium with pH values ​​of 6.0, 5.8, 5.6, 5.5, 5.4, 5.3, and 5.2, respectively. After culturing at 30°C and 200 rpm for 24 h, the OD values ​​were measured. 600 Each series of experiments consisted of 3 parallel experiments.

[0158] The results showed that there was no significant difference in acid resistance between WT and WT / pXMJ19. Compared with the wild-type strain and the control strain, NS-Cg had a significant growth advantage in the medium with pH 6.0-pH 5.2, indicating that its ability to resist acid stress was enhanced ( Figure 4 Among them, at pH 5.5, the OD600 of NS-Cg after 24 h of culture was 4.3, which was 1.95 times that of WT (OD600 was 2.2) and 1.99 times that of WT / pXMJ19 (OD600 was 2.16).

[0159] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A tool for random mutation of the genome of Corynebacterium glutamicum, characterized in that The tool comprises a plasmid vector skeleton, on which are assembled and connected: a nucleotide sequence encoding DNA adenine deaminase and a nucleotide sequence encoding Corynebacterium glutamicum DNA helicase.

2. The tool for random mutation of the Corynebacterium glutamicum genome according to claim 1, characterized in that The DNA adenine deaminase includes any one of TadA8e, TadA7.10, TadA8.17, TadA8.20, and TadA9; The DNA helicase includes any one of Cgl0025, Cgl0141, Cgl0776, Cgl0779, Cgl0846, Cgl0854, Cgl0894, Cgl1156, Cgl1326, Cgl1927, and Cgl2519; Preferably, the DNA adenine deaminase is TadA8e; the amino acid sequence of the DNA adenine deaminase TadA8e is shown in SEQ ID NO.1; Preferably, the DNA helicase is Cgl2519; the amino acid sequence of the DNA helicase Cgl2519 is shown in SEQ ID NO.2; Preferably, the DNA adenine deaminase TadA8e is fused to the C-terminal endometrium of the DNA helicase Cgl2519 via a flexible linker.

3. The Corynebacterium glutamicum genome random mutation tool according to claim 1 or 2, characterized in that The amino acid sequence of the linker is shown in SEQ ID NO.

3.

4. The tool for random mutation of the Corynebacterium glutamicum genome according to any one of claims 1 to 3, characterized in that The plasmid vector is selected from any one of pXMJ19, pDXW10 and pEC-XK99E.

5. A recombinant Corynebacterium glutamicum, characterized in that The method is obtained by transforming Corynebacterium glutamicum with the random mutation tool for the Corynebacterium glutamicum genome according to any one of claims 1 to 4.

6. A method for improving the acid resistance, high temperature resistance, oxidation resistance and / or osmotic pressure resistance of Corynebacterium glutamicum, characterized in that: The method comprises transforming Corynebacterium glutamicum with the Corynebacterium glutamicum genome random mutation tool according to any one of claims 1 to 4.

7. Use of the tool for random mutation of the Corynebacterium glutamicum genome according to any one of claims 1 to 4 in improving the robustness and / or biosynthetic efficiency of Corynebacterium glutamicum, or in screening for strains with high robustness and / or biosynthetic efficiency.

8. A method for screening Corynebacterium glutamicum with high acid resistance, characterized in that: The method comprises the following steps: transforming Corynebacterium glutamicum with the random mutation tool for the Corynebacterium glutamicum genome according to any one of claims 1 to 4 to obtain recombinant Corynebacterium glutamicum; and continuously subculturing the obtained recombinant Corynebacterium glutamicum under acidic conditions.

9. A method for increasing the rifampicin-resistant mutation rate of Corynebacterium glutamicum, characterized in that: The method comprises transforming Corynebacterium glutamicum with the Corynebacterium glutamicum genome random mutation tool according to any one of claims 1 to 4.

10. The method for increasing the rifampicin-resistant mutation rate of Corynebacterium glutamicum according to claim 9, characterized in that: The method comprises fusing DNA adenine deaminase TadA8e and the C-terminal endometrium of DNA helicase Cgl2519 via a flexible linker to obtain a fusion protein; After the fusion protein is connected to the pXMJ19 plasmid, a recombinant vector is obtained; and the obtained recombinant vector is introduced into Corynebacterium glutamicum.

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