DNA polymerase III mutant and application thereof

Abstract

The invention discloses a DNA polymerase III mutant and application thereof. The mutant comprises an amino acid sequence as shown in SEQ ID No.1. The mutant can be applied to improvement of host bacterium genome mutation frequency. Escherichia coli is taken as a starting strain, and efficient evolution helper plasmids are introduced, so that the host mutation frequency is improved; and combining with the tryptophan structural analogue 5MT competitive pressure directional screening to finally obtain the escherichia coli engineering strain with high tryptophan yield. The helper plasmid comprises a pBad24 plasmid vector skeleton and an engineered escherichia coli DNA polymerase III danQ nucleotide fragment. The high-efficiency evolution helper plasmid provided by the invention can significantly improve the mutation rate of a host genome, can greatly shorten the traditional mutagenesis period by combining with 5MT directional screening, can efficiently screen high-yield tryptophan engineering strains, and has important application value.

Description

Technical Field

[0001] The invention relates to a DNA polymerase mutant and an application thereof, in particular to a DNA polymerase III mutant derived from Escherichia coli and an application thereof in increasing the mutation frequency of a host bacterial genome and in evolutionary screening of high-yield strains. Background Art

[0002] L-tryptophan is an essential amino acid that humans and animals rely on for external intake. Since its first discovery and isolation in 1901, L-tryptophan, as an important metabolite in the body, has been proven to play a key role in the metabolic regulation of protein synthesis, fat metabolism and other substances.

[0003] With the development of biotechnology, microbial fermentation has become the preferred technology for the industrial production of L-tryptophan due to its low cost, environmental protection, high cell density and high production efficiency. Since wild-type Escherichia coli synthesizes tryptophan to meet its own growth needs, and there are many feedback inhibition nodes in the tryptophan synthesis pathway, it is difficult for tryptophan to accumulate in large quantities in Escherichia coli. Therefore, existing tryptophan production strains generally have the problem of weak tryptophan synthesis ability.

[0004] In order to improve the tryptophan synthesis ability of the strain, the strain can be continuously screened by adaptive evolution in order to obtain a high-yield strain. Traditional adaptive laboratory evolution usually requires a long evolutionary cycle, mainly because the mutation frequency of the E. coli genome is low under natural conditions. The dnaQ gene encodes the 3′ exonuclease-ε subunit of DNA polymerase III with proofreading function, which is a key gene responsible for the fidelity replication of the E. coli genome. Weakening its function may lead to an increase in the mutation frequency of the E. coli genome. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a DNA polymerase III mutant to solve the problem of how to competitively inhibit the function of wild-type DNA polymerase III. Another purpose of the present invention is to propose an application of the above-mentioned DNA polymerase III mutant in improving the mutation frequency of the host bacterial genome to solve the problem of how to improve the mutation frequency of the host bacterial genome. The third purpose of the present invention is to provide an application of the above-mentioned DNA polymerase III mutant in an adaptive evolution strain to solve the problem of how to evolve existing engineered bacteria to obtain high-tryptophan-producing engineered bacteria. The fourth purpose of the present invention is to provide a plasmid containing the gene of the above-mentioned DNA polymerase III mutant to solve the problem of how to amplify or express the DNA polymerase III mutant.

[0006] Technical solution: The DNA polymerase III mutant described in the present invention comprises the amino acid sequence shown in SEQ ID No.1.

[0007] The mutant is obtained by double-site mutation of the amino acid sequence of wild-type DNA polymerase III, and the specific mutation sites include mutation of leucine at position 73 to tryptophan and mutation of alanine at position 164 to valine. Mutation of the two amino acid sites will lead to the loss of proofreading activity of DNA polymerase III.

[0008] The second aspect of the present invention provides an application of the above-mentioned DNA polymerase III mutant in increasing the mutation frequency of the host bacterial genome.

[0009] Preferably, the host bacteria is a tryptophan-producing recombinant Escherichia coli.

[0010] Preferably, the Escherichia coli is one of the strains W3110, DH5α, and BL21.

[0011] In some embodiments, the specific method of the above application includes the following steps:

[0012] (1) cloning the wild-type Escherichia coli DNA polymerase III gene to obtain the dnaQ gene, and inserting the dnaQ gene into a vector to construct a first expression vector;

[0013] (2) using the first expression vector as a template, using mutant primers to perform PCR amplification of the mutant dnaQ gene, and obtaining a second expression vector containing the DNA polymerase III mutant gene after one-step cloning;

[0014] (3) Transforming the second expression vector into the host bacteria to express the DNA polymerase III mutant.

[0015] Excessive expression of non-functional DNA polymerase III in the host bacteria through the second expression vector will compete with the functional DNA polymerase III expressed in the genome, resulting in a decrease in the fidelity of the E. coli genome replication process, thereby greatly increasing the mutation frequency of the strain genome.

[0016] Preferably, in step (1), the cloning method of the dnaQ gene comprises: using the Escherichia coli genome as a template and the nucleic acid sequence shown in SEQ ID No.3-4 as a primer to perform PCR amplification to obtain the dnaQ gene; the method of inserting the dnaQ gene into the vector comprises: double-enzyme digestion of the plasmid to obtain a linearized vector fragment, and connecting the dnaQ gene to the linearized vector fragment to obtain a circular first expression vector.

[0017] Preferably, in step (1), the vector is a pBad24 plasmid, and its nucleotide sequence is shown in SEQ ID NO.11. The promoter in the plasmid is ParaBAD, and arabinose is required to induce the expression of the dnaQ gene. Other elements with transcription initiation function can be used as less preferred alternatives. The nucleotide sequence of the ParaBAD promoter is shown in SEQ ID NO.12.

[0018] Preferably, in step (2), the method of using mutant primers for PCR amplification is: using the first primer pair shown in SEQ ID No.5-6 to amplify the first expression vector to obtain the third expression vector, and then using the second primer pair shown in SEQ ID No.7-8 to amplify the third expression vector to obtain the second expression vector;

[0019] Alternatively, the first primer pair shown in SEQ ID No.7-8 is used to amplify the first expression vector to obtain the third expression vector, and then the second primer pair shown in SEQ ID No.5-6 is used to amplify the third expression vector to obtain the second expression vector.

[0020] The third aspect of the present invention provides a use of the above-mentioned DNA polymerase III mutant in adaptive evolution strains.

[0021] In some embodiments, the specific method of the above application includes:

[0022] The tryptophan structural analog 5-methyltryptophan (5-MT) was used to rapidly screen for highly tolerant strains, and ultimately a high-yield tryptophan engineered bacterium was obtained.

[0023] As a structural analog of tryptophan, 5-MT can specifically and competitively bind to key enzymes in the tryptophan synthesis pathway, thereby inhibiting the synthesis of tryptophan and causing strain growth difficulties. This inhibitory effect allows only strains with high tolerance to 5-MT to grow on a medium containing 5-MT, and these strains can also tolerate 5-MT structural analogs—tryptophan, thereby achieving rapid screening of high-yield tryptophan engineering bacteria.

[0024] Preferably, the working concentration of 5-MT in the method for efficient positive screening of tryptophan-producing strains is 2 g / L.

[0025] The present invention intends to start from the model strain W3310, utilize genetic engineering technology to maximize the mutation frequency of the host genome, and then combine the competitive screening pressure of structural analogs to globally regulate the tryptophan synthesis pathway, efficiently positively screen high-tolerance biological chassis, and realize efficient biosynthesis of tryptophan.

[0026] The fourth aspect of the present invention provides a plasmid containing the gene of the above-mentioned DNA polymerase III mutant.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0028] The efficient evolution auxiliary plasmid PQM (i.e., the second expression vector) constructed by the present invention can express a large amount of non-functional DNA polymerase III, which competes with the functional DNA polymerase III expressed by the host bacterial genome, resulting in a significant increase in the mutation frequency of the Escherichia coli genome replication process, thereby achieving efficient evolution of the strain. The evolved strain is cultured under conditions containing a high concentration of tryptophan structural analog 5-MT, and only high-tolerance tryptophan strains can grow normally, thereby achieving the screening of high-yield tryptophan engineering bacteria.

[0029] The method of combining efficient evolution strains with structural analogue pressure screening of the present invention not only shortens the laboratory adaptive evolution strain cycle, but also applies it to the breeding of excellent biological chassis for tryptophan, which is of great significance for the industrial application of green biosynthesis of tryptophan. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A schematic diagram of the structure of the efficient evolution auxiliary plasmid PQM provided by the present invention;

[0031] Figure 2 The tryptophan production of the PQM 1-8 engineered bacteria in the present invention;

[0032] Figure 3 This is the tryptophan production status of the PQM 2a-PQM 2j engineered bacteria in the present invention. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0034] Unless otherwise specified in the examples, all methods used are conventional methods and all reagents are available from commercial sources.

[0035] The molecular biology experimental techniques used in the following examples include PCR amplification, vector construction, plasmid extraction, DNA fragment ligation, gel electrophoresis and other technical operations. For details, please refer to "Molecular Cloning Experiment Guide" (3rd edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Translated by Huang Peitang et al., 2002, Beijing: Science Press).

[0036] The pBad24 plasmid involved in the present invention is from the collection of Huaibei Mining Green Chemical New Materials Research Institute.

[0037] Example 1: A DNA polymerase III mutant is obtained by double-site mutation of the amino acid sequence of wild-type DNA polymerase III, wherein the specific mutation sites include mutation of leucine at position 73 to tryptophan and mutation of alanine at position 164 to valine.

[0038] The amino acid sequence of the mutant is shown in SEQ ID No.1:

[0039] MSTAITRQIVLDTETTGMNQIGAHYEGHKIIEIGAVEVVNRRRLTGNNFHVYLKPD RLVDPEAFGVHGIADEF W LDKPTFAEVADEFMDYIRGAELVIHNAAFDIGFMDYEFSLLKRDIPKTNTFCKVTDSLAVARKMFPGKRNSLDALCARYEIDNSKRTLHG V LLDAQILAEVYLAMTGGQTSMAFAMEGETQQQQGEATIQRIVRQASKLRVVFATDEEIAAHEARLDLVQKKGGSCLWRA*

[0040] The underlined amino acids are mutated.

[0041] The gene sequence of wild-type DNA polymerase III is shown in SEQ ID No. 2:

[0042] ATGAGCACTGCAATTACACGCCAGATCGTTCTCGATACCGAAACCACCGGTATGAACCAGATTGGTGCGCACTATGAAGGCCACAAGATCATTGAGATTGGTGCCGTTGAAGTGGTGAACCGTCGCCTGACGGGCAATAACTTCCATGTTTATCTCAAACCCGATCGGCTGGTGGATCCGGAA GCCTTTGGCGTACATGGTATTGCCGATGAATTTTTGCTCGATAAGCCCACGTTTGCCGAAGTAGCCGATGAGTTCATGGACTATATTCGCGGCGCGGAGTTGGTGATCCATAACGCAGCGTTCGATATCGGCTTTATGGACTACGAGTTTTCGTTGCTTAAGCGCGATATTCCGAAGACCAAT ACTTTCTGTAAGGTCACCGATAGCCTTGCGGTGGCGAGGAAAATGTTTCCCGGTAAGCGCAACAGCCTCGATGCGTTATGTGCTCGCTACGAAATAGATAACAGTAAACGAACGCTGCACGGGGCATTACTCGATGCCCAGATCCTTGCGGAAGTTTATCTGGCGATGACCGGTGGTCAAACG TCGATGGCTTTTGCGATGGAAGGAGACACAACAGCAACAAGGTGAAGCAACAATTCAGCGCATTGTACGTCAGGCAAGTAAGTTACGCGTTGTTTTTGCGACAGATGAAGAGATTGCAGCTCATGAAGCCCGTCTCGATCTGGTGCAGAAGAAAGGCGGAAGTTGCCTCTGGCGAGCATAA

[0043] The method for using the above DNA polymerase III mutant to increase the mutation frequency of the host bacterial genome is as follows:

[0044] (1) Design cloning primers and mutation primers for the dnaQ gene, the sequences of which are shown in Table 1:

[0045] Table 1. Primers used in the construction of pBad24-dnaQ+MS and their corresponding sequences

[0046]

[0047] (2) Using the E. coli W3110 genome as a template and dnaQ-F and dnaQ-R as primers, PCR amplification was performed to obtain the dnaQ product. After the band size was confirmed by gel electrophoresis, a gel recovery kit (Shanghai Sangon Biotechnology Co., Ltd.) was used to recover the dnaQ fragment carrying the overlapping sequence of the pBad24 vector, the sequence of which is shown in SEQ ID No. 9:

[0048] TGGGCTAGCAGGAGGAATTC ATGAGCACTGCAATTACACGCCAGATCGTTCTCGAT

[0049] ACCGAAACCACCGGTATGAACCAGATTGGTGCGCACTATGAAGGCCACAAGATCA

[0050] TTGAGATTGGTGCCGTTGAAGTGGTGAACCGTCGCCTGACGGGCAATAACTTCCAT

[0051] GTTTATCTCAAACCCGATCGGCTGGTGGATCCGGAAGCCTTTGGCGTACATGGTATT

[0052] GCCGATGAATTTTTGCTCGATAAGCCCACGTTTGCCGAAGTAGCCGATGAGTTCAT

[0053] GGACTATATTCGCGGCGCGGAGTTGGTGATCCATAACGCAGCGTTCGATATCGGCTT

[0054] TATGGACTACGAGTTTTCGTTGCTTAAGCGCGATATTCCGAAGACCAATACTTTCTG

[0055] TAAGGTCACCGATAGCCTTGCGGTGGCGAGGAAAATGTTTCCCGGTAAGCGCAAC

[0056] AGCCTCGATGCGTTATGTGCTCGCTACGAAATAGATAACAGTAAACGAACGCTGCA

[0057] CGGGGCATTACTCGATGCCCAGATCCTTGCGGAAGTTTATCTGGCGATGACCGGTG

[0058] GTCAAACGTCGATGGCTTTTGCGATGGAAGGAGAGACACAACAGCAACAAGGTG

[0059] AAGCAACAATTCAGCGCATTGTACGTCAGGCAAGTAAGTTACGCGTTGTTTTTGCG

[0060] ACAGATGAAGAGATTGCAGCTCATGAAGCCCGTCTCGATCTGGTGCAGAAGAAAG

[0061] GCGGAAGTTGCCTCTGGCGAGCATAA AAGCTTGGCTGTTTTGGCGG

[0062] The underlined part is the overlap segment.

[0063] The PCR reaction system and procedure are shown in Table 2 and Table 3:

[0064] Table 2. DNAQ amplification reaction system

[0065]

[0066] Table 3. DNAQ amplification reaction program

[0067]

[0068] (3) The pBad24 plasmid was double-digested with EcoRⅠ and HindⅢ (Vazyme Biotech Co., Ltd) restriction endonucleases. After the band size was confirmed by gel electrophoresis, the linearized pBad24 vector fragment was recovered using a gel recovery kit;

[0069] (4) The dnaQ fragment and the linearized pBad24 vector fragment were connected by Gibson assembly technology. For specific operations, see the instructions of ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co., Ltd). After connection, the circular plasmid pBad24-dnaQ was obtained. The plasmid was transferred into E. coli DH5α by chemical transformation and cultured overnight at 37°C. A single colony was picked for colony PCR verification. The strains that were initially identified successfully were sent to Shanghai Sangon Biotechnology Co., Ltd. for further sequencing confirmation. After the strains that were successfully sequenced were cultured, the pBad24-dnaQ plasmid was extracted using a plasmid extraction kit (Shanghai Sangon Biotechnology Co., Ltd.) for use.

[0070] (5) Using the pBad24-dnaQ plasmid as a template, PCR amplification was performed using the primer pair dnaQ(L73W)-F / dnaQ(L73W)-R to obtain the pBad24-dnaQ(L73W) fragment with overlapping fragments at both ends. After one-step cloning, it was transferred into E. coli DH5α and identified to obtain the plasmid pBad24-dnaQ(L73W). The specific operations of one-step cloning, transformation, and positive clone identification were the same as those in step (4). The PCR reaction system and procedure are shown in Tables 4 and 5.

[0071] Table 4. Construction of pBad24-dnaQ (L73W) plasmid reaction system

[0072]

[0073] Table 5. Reaction procedure for constructing pBad24-dnaQ(L73W) plasmid

[0074]

[0075] (6) Using the pBad24-dnaQ(L72W) plasmid as a template, the primer pair dnaQ(A164V)-F / dnaQ(A164V)-R was used to perform PCR amplification to obtain a dnaQ double mutation fragment with overlaps at both ends. After one-step cloning, it was transferred into E. coli DH5α. After identification, the efficient evolution auxiliary plasmid vector pBad24-dnaQ+MS (abbreviated as PQM) was obtained. The specific operations of one-step cloning, transformation, and positive clone identification were the same as those in step (3). The PCR reaction system and procedure are shown in Tables 6 and 7. The schematic diagram of the efficient evolution recombinant plasmid pBad24-dnaQ+MS is shown in Figure 6. Figure 1 As shown, it includes key elements such as araC (arabinose repressor protein), araBAD (arabinose inducible promoter), and danQ+MS (engineered danQ gene, leucine at position 73 mutated to tryptophan, and alanine at position 164 mutated to valine). The sequence of the danQ+MS element is shown in S EQ ID No.10:

[0076] ATGAGCACTGCAATTACACGCCAGATCGTTCTCGATACCGAAACCACCGGTATGAA

[0077] CCAGATTGGTGCGCACTATGAAGGCCACAAGATCATTGAGATTGGTGCCGTTGAAG

[0078] TGGTGAACCGTCGCCTGACGGGCAATAACTTCCATGTTTATCTCAAACCCGATCGG

[0079] CTGGTGGATCCGGAAGCCTTTGGCGTACATGGTATTGCCGATGAATTT TGG CTCGAT

[0080] AAGCCCACGTTTGCCGAAGTAGCCGATGAGTTCATGGACTATATTCGCGGCGCGGA

[0081] GTTGGTGATCCATAACGCAGCGTTCGATATCGGCTTTATGGACTACGAGTTTTCGTT

[0082] GCTTAAGCGCGATATTCCGAAGACCAATACTTTCTGTAAGGTCACCGATAGCCTTG

[0083] CGGTGGCGAGGAAAATGTTTCCCGGTAAGCGCAACAGCCTCGATGCGTTATGTGCT

[0084] CGCTACGAAATAGATAACAGTAAACGAACGCTGCACGGG GTA TTACTCGATGCCCA

[0085] GATCCTTGCGGAAGTTTATCTGGCGATGACCGGTGGTCAAACGTCGATGGCTTTTG

[0086] CGATGGAAGGAGAGACACAACAGCAACAAGGTGAAGCAACAATTCAGCGCATTG

[0087] TACGTCAGGCAAGTAAGTTACGCGTTGTTTTTGCGACAGATGAAGAGATTGCAGCT

[0088] CATGAAGCCCGTCTCGATCTGGTGCAGAAGAAAGGCGGAAGTTGCCTCTGGCGAG

[0089] CATAA

[0090] The underlined bases are mutated bases.

[0091] Table 6. Construction of PQM plasmid reaction system

[0092]

[0093] Table 7. Reaction procedures for constructing PQM plasmids

[0094]

[0095] (7) Construction of Escherichia coli efficient evolution engineering bacteria: The starting strain is Escherichia coli W3310 (Coli Genetic Stock Center strain (CGSC) No. 4474), and competent Escherichia coli W3110 is prepared. The plasmid PQM is introduced into the competent Escherichia coli W3110 by chemical transformation. The specific operations of transformation and positive clone identification are the same as step (3) in Example 1, and finally an Escherichia coli W3110 strain carrying the PQM plasmid is obtained. The PQM plasmid can synthesize non-functional DNA polymerase III under the regulation of arabinose, and compete with the functional DNA polymerase III expressed in the genome, resulting in a significant increase in the mutation frequency of the Escherichia coli genome replication process, thereby achieving efficient evolution of the strain.

[0096] Example 2: The screening method for high tryptophan production engineering bacteria is as follows:

[0097] (1) Take the E. coli W3110 strain carrying the PQM plasmid prepared in Example 1, inoculate it in LB medium overnight, transfer it to 8 shake flasks in LB medium supplemented with 0.5% arabinose at a 1% inoculum, and wait until the strain grows to OD 600 The cells were collected by centrifugation at a concentration between 0.6 and 0.8.

[0098] (2) Dilute the cells with saline to OD 600 0.1, take 200 μL of the diluted bacterial solution and spread it on the screening LB plate, which is added with 2g / L 5MT. Only the mutant high-tolerance strains can grow on the plate. Incubate in a 37℃ incubator for 15 hours, and select one strain with good growth from each of the 8 plates and name it PQM 1-8.

[0099] (3) PQM 1-8 engineered bacteria were used for shake flask fermentation. The fermentation medium composition was as follows: KH2PO4 3.4 g / L, K2HPO4·3H2O 11.4 g / L, (NH4)2SO4 6 g / L, trisodium citrate 3 g / L, MgSO4·7H2O 3 g / L, FeSO4·7H2O0.113 g / L, Glucose 20 g / L and trace elements 3 mL / L (CoCl·6H2O 4 g / L, CuSO4·5H2O0.6 g / L, MnSO4·H2O 4.5 g / L, ZnSO4 6.4 g / L). Specifically, single colonies were inoculated into test tubes containing LB medium, and cultured overnight at 37°C and 180 rpm to prepare seed solution. 1 mL of seed solution was inoculated into a 250 mL shake flask containing 20 mL of fermentation medium. The culture was then shaken at 30°C and 180 rpm for 48 hours, and the tryptophan production was detected by HPLC (see Krcmova L, Solichova D, Melichar B, et al. Determination of neopterin, kynurenine, tryptophan and creatinine in human serum by high throuput HPLC [J]. Talanta, 2011, 85 (3): 1466-1471. DOI: 10.1016 / j.talanta.2011.06.027.). The tryptophan production by PQM 1-8 engineered bacteria was as follows: Figure 2 As shown, strain PQM 2 showed the highest tryptophan biosynthesis ability, with a yield of 2.31 g / L.

[0100] (4) Subculture and taming of engineered bacteria to stabilize their tryptophan biosynthesis capacity

[0101] Take strain PQM 2 for subculture and domestication to stabilize its tryptophan synthesis ability. Specifically, streak PQM 2 on an LB plate supplemented with 2g / L5-MT, culture overnight at 37°C, pick a single colony and inoculate it into LB liquid culture medium, and culture overnight at 37°C and 180rpm. Inoculate in an LB shake flask supplemented with 2g / L 5-MT at a 5% inoculation rate. When the biomass of the engineered bacteria enters the plateau phase, re-inoculate in a new LB shake flask supplemented with 2g / L 5-MT at a 5% inoculation rate, record it as one generation, and subculture 10 generations. Take a sample of the 10th generation of engineered bacteria and streak it on an LB solid plate for overnight culture, isolate a single colony, and obtain an individual domesticated strain.

[0102] Eight subcultured PQM 2a-PQM 2h strains were used for shake flask fermentation to evaluate their tryptophan synthesis ability. The specific operation was the same as in Example 3. The fermentation production of tryptophan by the domesticated engineering bacteria was as follows: Figure 3Finally, the tryptophan high-yield engineered bacteria PQM 2h with stable traits was obtained, and the yield reached a maximum of 2.42g / L.

[0103] Comparative Example 1: The rest is the same as Example 1, except that:

[0104] Only the leucine at position 73 was mutated to tryptophan, and the mutation at position 164 was not performed.

[0105] The method of Example 2 was used to screen high tryptophan-producing engineered bacteria.

[0106] Comparative Example 2: The rest is the same as Example 1, except that:

[0107] Only the alanine at position 164 was mutated to valine, and the mutation operation at position 73 was not performed.

[0108] The method of Example 2 was used to screen high tryptophan-producing engineered bacteria.

[0109] Comparative Example 3: The rest is the same as Example 1, except that:

[0110] Instead of performing the mutation operation at position 73, the phenylalanine at position 72 was mutated to tryptophan. The mutation primers are as follows:

[0111] dnaQ(F72W)-F:ggtattgccgatgaaTGGttgctcg

[0112] dnaQ(F72W)-R:cgagcaaCCAttcatcggcaatacc

[0113] The annealing temperature is 61°C.

[0114] The method of Example 2 was used to screen high tryptophan-producing engineered bacteria.

[0115] Comparative Example 4: The rest is the same as Example 1, except that:

[0116] Instead of performing the mutation operation at position 164, the glycine at position 163 was mutated to valine. The mutation primers are as follows:

[0117] dnaQ(G163V)-F:gaacgctgcacGTAgcattactcg

[0118] dnaQ(G163V)-R:cgagtaatgcTACgtgcagcgttc

[0119] The annealing temperature is 60°C.

[0120] The method of Example 2 was used to screen high tryptophan-producing engineered bacteria.

[0121] The genome mutation rate and tryptophan production of the wild-type W3110 strain and the recombinant bacteria constructed in Example 1 and Comparative Examples 1-3 were detected. The detection method was as follows: the strains carrying various plasmids were cultured in LB liquid culture medium at 37°C overnight. An aliquot of the culture was inoculated on a solid LB plate containing 5-MT and incubated in a 37°C incubator for 24 hours. The colonies formed on the added 5MT plate were counted as resistant mutants. An aliquot of the culture was inoculated on an LB plate and incubated at 37°C for 12 hours. The number of colonies formed on the plate was recorded as the number of viable cells. To ensure data accuracy, no less than 10 plates were included in each group for a single experiment. The mutation frequency calculation formula is: number of resistant cells / number of viable cells. The results are as follows:

[0122] Table 1 Effects of different mutation sites on genome mutation rate and tryptophan production

[0123]

[0124]

[0125] As can be seen from the results in Table 1, the mutants formed by single-site mutations did not significantly increase the mutation frequency of the E. coli genome, resulting in a significant reduction in the possibility of screening for high-yield strains. In the case of double-site mutations, changes in any mutation site could not cause the mutant to produce a competitive inhibitory effect on wild-type DNA polymerase III, which also caused the genome mutation frequency to remain at a low level, with no significant difference from the wild-type control group.

[0126] Only when the amino acids at positions 73 and 164 mutate at the same time can the mutant be formed to competitively inhibit the wild-type DNA polymerase III, thereby significantly increasing the mutation frequency of the Escherichia coli genome and greatly increasing the possibility of screening and obtaining high-yield strains, which is beneficial to the adaptive evolution of the strain.

Claims

1. A DNA polymerase III mutant, characterized in that: It comprises the amino acid sequence shown in SEQ ID No.

1.

2. Use of the DNA polymerase III mutant according to claim 1 in increasing the mutation frequency of the host bacterial genome.

3. The use according to claim 2, characterized in that: The host bacteria is a tryptophan-producing recombinant Escherichia coli.

4. The use according to claim 3, characterized in that: The Escherichia coli is one of the strains W3110, DH5α and BL21.

5. The use according to claim 2, characterized in that: The steps include: (1) cloning the wild-type Escherichia coli DNA polymerase III gene to obtain the dnaQ gene, and inserting the dnaQ gene into a vector to construct a first expression vector; (2) using the first expression vector as a template, using mutant primers to perform PCR amplification of the mutant dnaQ gene, and obtaining a second expression vector containing the DNA polymerase III mutant gene after one-step cloning; (3) Transforming the second expression vector into the host bacteria to express the DNA polymerase III mutant.

6. The use according to claim 5, characterized in that: In step (1), the cloning method of the dnaQ gene includes: using the Escherichia coli genome as a template and the nucleic acid sequence shown in SEQ ID No.3-4 as a primer to perform PCR amplification to obtain the dnaQ gene; the method of inserting the dnaQ gene into the vector includes: double-enzyme digestion of the plasmid to obtain a linearized vector fragment, and connecting the dnaQ gene to the linearized vector fragment to obtain a circular first expression vector.

7. The use according to claim 5, characterized in that: In step (1), the vector is pBad24.

8. The use according to claim 5, characterized in that: In step (2), the method of using mutant primers for PCR amplification is: using the first primer pair shown in SEQ ID No.5-6 to amplify the first expression vector to obtain the third expression vector, and then using the second primer pair shown in SEQ ID No.7-8 to amplify the third expression vector to obtain the second expression vector; Alternatively, the first primer pair shown in SEQ ID No.7-8 is used to amplify the first expression vector to obtain the third expression vector, and then the second primer pair shown in SEQ ID No.5-6 is used to amplify the third expression vector to obtain the second expression vector.

9. Use of the DNA polymerase III mutant according to claim 1 in adaptive evolution strains.

10. A plasmid containing the gene of the DNA polymerase III mutant according to claim 1.

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