A mutant of DNA polymerase iii and its use

Abstract

The application discloses a DNA polymerase III mutant and application thereof. The mutant contains an amino acid sequence shown in SEQ ID No. 1, and the mutant can be applied to improving the mutation frequency of a host strain genome. In the application, the Escherichia coli is used as a starting strain, a high-efficiency evolution auxiliary plasmid is introduced, and the mutation frequency of the host is improved; combined with competitive pressure directional screening of a tryptophan structural analog 5MT, finally, the Escherichia coli engineering strain with high yield of tryptophan is obtained. The auxiliary plasmid comprises a pBad24 plasmid vector skeleton and an engineered Escherichia coli DNA polymerase III danQ nucleotide fragment. The high-efficiency evolution auxiliary plasmid provided by the application can significantly improve the mutation rate of the host genome, combined with the directional screening of 5MT, the traditional mutagenesis cycle can be greatly shortened, the engineering strain with high yield of tryptophan can be efficiently screened, and the application has important application value.

Description

TECHNICAL FIELD

[0001] The present application relates to a DNA polymerase mutant and its application, in particular to a DNA polymerase III mutant from Escherichia coli and its application in improving the genome mutation frequency of host bacteria and screening high-yield strains. BACKGROUND

[0002] L-tryptophan is an essential amino acid for humans and animals, which is dependent on external intake. Since it was first discovered and isolated in 1901, as an important metabolic product in vivo, the key role of L-tryptophan in protein synthesis, fat metabolism and the metabolic regulation of other substances has been proven.

[0003] With the development of biotechnology, microbial fermentation method has become the preferred technology for 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, the existing tryptophan-producing strains generally have weak tryptophan synthesis ability.

[0004] In order to improve the tryptophan synthesis ability of the strain, the strain can be continuously adapted and evolved to obtain a high-yield strain. Traditional adaptive laboratory evolution usually requires a long evolution period, mainly because the genome mutation frequency of Escherichia coli is low under natural conditions. The dnaQ gene encodes the 3' exonuclease-epsilon subunit of DNA polymerase III with proofreading function, which is a key gene responsible for the faithful replication of the genome of Escherichia coli. Weakening its function may increase the genome mutation frequency of Escherichia coli. SUMMARY

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

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

[0007] The mutant is obtained by double site mutation from 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. The mutation of the two amino acid sites will result in loss of correction activity of DNA polymerase III.

[0008] The second aspect of the present application provides a use of the above-mentioned DNA polymerase III mutant in improving the mutation frequency of the genome of a host bacterium.

[0009] Preferably, the host bacterium is recombinant Escherichia coli producing tryptophan.

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

[0011] In some embodiments, the specific method of the above-mentioned use comprises the following steps:

[0012] (1) cloning the wild type DNA polymerase III gene of Escherichia coli to obtain a 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 and using a mutation primer 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 bacterium to express the DNA polymerase III mutant.

[0015] By expressing the non-functional DNA polymerase III in the host bacterium through the second expression vector, competition with the functional DNA polymerase III expressed from the genome is caused, resulting in a decrease in fidelity during replication of the Escherichia coli genome, and thus the mutation frequency of the genome of the strain is greatly improved.

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

[0017] Preferably, in step (1), the vector is a pBad24 plasmid, and the nucleotide sequence thereof is shown in SEQ ID NO. 11. The promoter in the plasmid is ParaBAD, and the expression of the dnaQ gene needs to be induced by arabinose. Other elements having a transcription initiation function can be used as a suboptimal alternative. The nucleotide sequence of the ParaBAD promoter is shown in SEQ ID NO. 12.

[0018] Preferably, in step (2), the method for PCR amplification with mutation primers is as follows: the first expression vector is amplified with the first primer pair shown in SEQ ID No. 5-6 to obtain a third expression vector, and the third expression vector is amplified with the second primer pair shown in SEQ ID No. 7-8 to obtain the second expression vector.

[0019] Or the first expression vector is amplified with the first primer pair shown in SEQ ID No. 7-8 to obtain a third expression vector, and the third expression vector is amplified with the second primer pair shown in SEQ ID No. 5-6 to obtain the second expression vector.

[0020] The third aspect of the present application provides an application of the above-mentioned DNA polymerase III mutant in adaptive evolution of a bacterial strain.

[0021] In some embodiments, the specific method of the above-mentioned application comprises:

[0022] The tryptophan structural analog 5-methyltryptophan (5-MT) is used for directional and rapid screening of high-tolerance strains, and finally a high-yield tryptophan engineering bacterium is obtained.

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

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

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

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

[0027] Advantages: Compared with the prior art, the present application has the following obvious advantages:

[0028] The high-efficiency evolution auxiliary plasmid PQM (i.e., the second expression vector) constructed in the application can express a large amount of non-functional DNA polymerase III, which competes with the functional DNA polymerase III expressed by the genome of the host bacteria, so as to greatly increase the mutation frequency of the replication process of the genome of the Escherichia coli, and realize the high-efficiency evolution of the strain. The evolved strain is cultured under the condition of containing a high concentration of 5-MT, a structural analog of tryptophan, and only the strain with high tolerance to tryptophan can grow normally, so as to realize the screening of the high-yield tryptophan engineering bacteria.

[0029] The method for screening the high-efficiency evolution strain combined with the structural analog stress in the application not only shortens the period of laboratory adaptive evolution strain, but also applies it to the breeding of the excellent biological tryptophan chassis, and has important significance for the industrialized application of the green biosynthesis of tryptophan. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The structural schematic diagram of the high-efficiency evolution auxiliary plasmid PQM provided in the application is shown in the figure.

[0031] Figure 2 The tryptophan production of the PQM 1-8 engineering bacteria in the application is shown in the figure.

[0032] Figure 3 The tryptophan production of the PQM 2a-PQM 2j engineering bacteria in the application is shown in the figure. DETAILED DESCRIPTION

[0033] The technical solutions of the application will be further described below with reference to the drawings.

[0034] In the examples, the used methods are all conventional methods, and the reagents can be obtained from commercial channels.

[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, which can be specifically referred to in the Guide to Molecular Cloning Experiment (third edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peitang et al, 2002, Beijing: Science Press).

[0036] The pBad24 plasmid involved in the application is derived from the collection of Huaibei Mining Green Chemical New Material 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, and 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] MSTAITRQIVLDTETTGMNQIGAHYEGHKIIEIGAVEVVNRRLTGNNFHVYLKPD RLVDPEAFGVHGIADEF W LDKPTFAEVADEFMDYIRGAELVIHNAAFDIGFMDYEFSLLKRDIPKTNTFCKVTDSLAVARKMFPGKRNSLDALCARYEIDNSKRTLHG V LLDAQILAEVYLAMTGGQTSMAFAMEGETQQQQGEATIQRIVRQASKLRVVFATDEEIAAHEARLDLVQKKGGSCLWRA*

[0040] wherein the underlined is the mutated amino acid.

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

[0042] ATGAGCACTGCAATTACACGCCAGATCGTTCTCGATACCGAAACCACCGGTATGAACCAGATTGGTGCGCACTATGAAGGCCACAAGATCATTGAGATTGGTGCCGTTGAAGTGGTGAACCGTCGCCTGACGGGCAATAACTTCCATGTTTATCTCAAACCCGATCGGCTGGTGGATCCGGAAGCCTTTGGCGTACATGGTATTGCCGATGAATTTTTGCTCGATAAGCCCACGTTTGCCGAAGTAGCCGATGAGTTCATGGACTATATTCGCGGCGCGGAGTTGGTGATCCATAACGCAGCGTTCGATATCGGCTTTATGGACTACGAGTTTTCGTTGCTTAAGCGCGATATTCCGAAGACCAATACTTTCTGTAAGGTCACCGATAGCCTTGCGGTGGCGAGGAAAATGTTTCCCGGTAAGCGCAACAGCCTCGATGCGTTATGTGCTCGCTACGAAATAGATAACAGTAAACGAACGCTGCACGGGGCATTACTCGATGCCCAGATCCTTGCGGAAGTTTATCTGGCGATGACCGGTGGTCAAACGTCGATGGCTTTTGCGATGGAAGGAGAGACACAACAGCAACAAGGTGAAGCAACAATTCAGCGCATTGTACGTCAGGCAAGTAAGTTACGCGTTGTTTTTGCGACAGATGAAGAGATTGCAGCTCATGAAGCCCGTCTCGATCTGGTGCAGAAGAAAGGCGGAAGTTGCCTCTGGCGAGCATAA

[0043] The method for increasing the mutation frequency of the genome of a host strain using the above-mentioned mutant of DNA polymerase III is as follows:

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

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

[0046]

[0047] (2) The dnaQ product was obtained by PCR amplification using the E. coli W3110 genome as a template and dnaQ-F and dnaQ-R as primers. After confirming the band size by gel electrophoresis, the dnaQ fragment carrying the pBad24 vector overlapping sequence was recovered using a gel recovery kit (Shanghai Biotechnology Engineering Co., Ltd.), and its sequence is shown as 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] GCGATGGAAGGAGAGACACAACAGCAACAAGGTG GCGAAGTTGCCTCTGGCGAGCATAA

[0059] GCGATGGAAGGAGAGACACAACAGCAACAAGGTG GCGAAGTTGCCTCTGGCGAGCATAA

[0060] GCGATGGAAGGAGAGACACAACAGCAACAAGGTG GCGAAGTTGCCTCTGGCGAGCATAA

[0061] GCGATGGAAGGAGAGACACAACAGCAACAAGGTG GCGAAGTTGCCTCTGGCGAGCATAA AAGCTTGGCTGTTTTGGCGG

[0062] Wherein, the underlined is the overlap fragment.

[0063] PCR reaction system and procedure see Table 2 and Table 3:

[0064] Table 2. Amplification of dnaQ reaction system

[0065]

[0066] Table 3. Amplification of dnaQ reaction procedure

[0067]

[0068] (3) The pBad24 plasmid was double digested by EcoR I and Hind III (Vazyme Biotech Co., Ltd) restriction enzymes, and after gel electrophoresis confirmed that the band size was correct, the linearized pBad24 vector fragment was recovered using a gel recovery kit;

[0069] (4) Gibson assembly technology was used to connect the dnaQ fragment and the linearized pBad24 vector fragment, and the specific operation was seen in the ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co., Ltd) instruction manual. After connection, the circular plasmid pBad24-dnaQ was obtained. The plasmid was transformed into E. coli DH5α using chemical transformation, and cultured at 37°C overnight. Single colonies were picked for colony PCR verification, and then the strains preliminarily identified successfully were sent to Shanghai Shengong Bioengineering Co., Ltd. for further sequencing confirmation. The successfully sequenced strain was cultured and the pBad24-dnaQ plasmid was extracted using a plasmid extraction kit (Shanghai Shengong Bioengineering Co., Ltd.) for standby.

[0070] (5) With pBad24-dnaQ plasmid as a template, primer pair dnaQ(L73W)-F / dnaQ(L73W)-R was used for PCR amplification to obtain pBad24-dnaQ(L73W) fragment with an overlap fragment at both ends, which was cloned into E. coli DH5α after one-step cloning, and pBad24-dnaQ(L73W) plasmid was obtained after identification. The one-step cloning, transformation, and positive clone identification were performed according to the procedures in step (4), and the PCR reaction system and procedure are shown in Table 4 and Table 5.

[0071] Table 4. Reaction system for constructing pBad24-dnaQ(L73W) plasmid

[0072]

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

[0074]

[0075] (6) With pBad24-dnaQ(L72W) plasmid as a template, primer pair dnaQ(A164V)-F / dnaQ(A164V)-R was used for PCR amplification to obtain dnaQ double mutant fragment with an overlap at both ends, which was cloned into E. coli DH5α after one-step cloning, and high-efficiency evolution auxiliary plasmid vector pBad24-dnaQ+MS (referred to as PQM) was obtained after identification. The one-step cloning, transformation, and positive clone identification were performed according to the procedures in step (3), and the PCR reaction system and procedure are shown in Table 6 and Table 7. The schematic diagram of high-efficiency evolution recombinant plasmid pBad24-dnaQ+MS is shown in Figure 1 , which includes araC (arabinose repressor protein), araBAD (arabinose inducible promoter), danQ+MS (engineered danQ gene, leucine at position 73 is mutated to tryptophan, alanine at position 164 is mutated to valine) key elements. The sequence of danQ+MS element is shown in SEQ 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] Wherein, the underlined is the mutant base.

[0091] Table 6. Reaction system for constructing PQM plasmid

[0092]

[0093] Table 7. Reaction procedure for constructing PQM plasmid

[0094]

[0095] (7) Construction of E. coli high-efficiency evolution engineering strain: The starting strain was E. coli W3110 (Coli Genetic Stock Center strain (CGSC) No. 4474). The competent E. coli W3110 was prepared, and the plasmid PQM was introduced into the competent E. coli W3110 by chemical transformation. The transformation and positive clone identification were performed according to the procedures in step (3) of Example 1. Finally, the E. coli W3110 strain carrying the PQM plasmid was obtained. The PQM plasmid can synthesize non-functional DNA polymerase III under the regulation of arabinose, which competes with the functional DNA polymerase III expressed by the genome, resulting in a significant increase in the mutation frequency of the replication process of the E. coli genome and realizing the high-efficiency evolution of the strain.

[0096] Example 2: The screening method of high-tryptophan-producing engineering strain is as follows:

[0097] (1) The E. coli W3110 strain carrying the PQM plasmid prepared in Example 1 was inoculated in LB medium and cultured overnight. The culture was inoculated in 8 flasks containing 0.5% arabinose LB medium at a 1% inoculation amount. When the strain grew to OD 600 between 0.6 and 0.8, the bacterial cells were collected by centrifugation.

[0098] (2) The bacterial cells were diluted with physiological saline to OD 600 0.1. 200 μL of the diluted bacterial solution was spread on a screening LB plate containing 2 g / L of 5MT. Only the mutated high-tolerance strain could grow on the plate. The plate was cultured in a 37°C incubator for 15 h. One strain with good growth state was selected from each of the 8 plates, and was named as PQM 1-8.

[0099] (3) Take the PQM 1-8 engineering bacteria for shake flask fermentation, and the fermentation medium composition is: 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, FeSO4·7H2O 0.113 g / L, glucose 20 g / L and trace elements 3 mL / L (CoCl·6H2O 4 g / L, CuSO4·5H2O 0.6 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 incubated at 37°C, 180 rpm overnight to prepare seed liquid. 1 mL of seed liquid was inoculated into a 250 mL shake flask containing 20 mL of fermentation medium. Then, it was cultured at 30°C, 180 rpm for 48 hours, and HPLC was used to detect the production of tryptophan (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. for details). The production of tryptophan synthesized by the PQM 1-8 engineering bacteria is shown in Table 2. Figure 2 As shown in Table 2, strain PQM 2 showed the highest tryptophan synthesis capacity, with a yield of 2.31 g / L.

[0100] (4) Passage domestication to stabilize the tryptophan synthesis capacity of the engineering bacteria

[0101] The strain PQM 2 was passaged and domesticated to stabilize its tryptophan synthesis capacity. Specifically, PQM 2 was streaked on LB plates with the addition of 2 g / L 5-MT, incubated at 37°C overnight, and then single colonies were picked and inoculated in LB liquid medium and incubated at 37°C, 180 rpm overnight. With a 5% inoculation amount, the engineering bacteria were inoculated in LB shake flasks with the addition of 2 g / L 5-MT, and when the biomass entered the platform phase, they were inoculated in new LB shake flasks with the addition of 2 g / L 5-MT with a 5% inoculation amount, which was recorded as one generation, and the passage was 10 generations. The 10th generation of engineering bacteria was sampled and streaked on LB solid plates for overnight incubation, and single colonies were isolated to obtain individual domesticated strains.

[0102] Eight passaged and domesticated PQM 2a-PQM 2h strains were taken for shake flask fermentation to evaluate their tryptophan synthesis capacity, and the specific operation was the same as in Example 3. The fermentation production of tryptophan by domesticated engineering bacteria is shown in Table 3. Figure 3Finally, the stable trait engineering strain PQM 2h with high yield of tryptophan was obtained, and the yield reached 2.42 g / L.

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

[0104] Only the leucine at position 73 was mutated to tryptophan, and no mutation operation was performed at position 164.

[0105] The high-yield tryptophan engineering strain was screened according to the method of Example 2.

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

[0107] Only the alanine at position 164 was mutated to valine, and no mutation operation was performed at position 73.

[0108] The high-yield tryptophan engineering strain was screened according to the method of Example 2.

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

[0110] No mutation operation was performed at position 73, but instead the phenylalanine at position 72 was mutated to tryptophan, and the mutation primer was as follows:

[0111] dnaQ(F72W)-F: ggtattgccgatgaaTGGttgctcg

[0112] dnaQ(F72W)-R: cgagcaaCCAttcatcggcaatacc

[0113] The annealing temperature was 61°C.

[0114] The high-yield tryptophan engineering strain was screened according to the method of Example 2.

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

[0116] No mutation operation was performed at position 164, but instead the glycine at position 163 was mutated to valine, and the mutation primer was as follows:

[0117] dnaQ(G163V)-F: gaacgctgcacGTAgcattactcg

[0118] dnaQ(G163V)-R: cgagtaatgcTACgtgcagcgttc

[0119] The annealing temperature was 60°C.

[0120] The high-yield tryptophan engineering strain was screened according to the method of Example 2.

[0121] The genomic mutation rate and tryptophan production of the wild-type W3110 strain and the recombinant strains constructed in Example 1 and Comparative Examples 1-3 were detected by the following method: the strains carrying various plasmids were cultured in LB liquid 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 h. The colonies formed on the plate with 5MT were counted as resistant mutants. An aliquot of the culture was inoculated on an LB plate and incubated at 37°C for 12 h, and 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 used for each group in a single experiment. The mutation frequency was calculated by the formula: number of resistant cells / number of viable cells. The results are as follows:

[0122] Table 1 Effect of different mutation sites on genomic mutation rate and tryptophan production

[0123]

[0124]

[0125] As can be seen from the results in Table 1, the mutants formed by single-site mutation do not significantly increase the genomic mutation frequency of E. coli, which greatly reduces the possibility of screening for high-yield strains. When double-site mutation occurs, the change of any mutation site cannot produce a competitive inhibition effect on the wild-type DNA polymerase III, which also leads to a low level of genomic mutation frequency, showing no significant difference from the wild-type control group.

[0126] Only when the mutants formed by simultaneous mutation of amino acids at positions 73 and 164 have the function of competitively inhibiting the wild-type DNA polymerase III, the genomic mutation frequency of E. coli is significantly increased, and the possibility of screening for high-yield strains is greatly increased, which is beneficial to the adaptive evolution of the strains.

Claims

1. A mutant of DNA polymerase III, characterized in that, The amino acid sequence of the mutant is shown as SEQ ID No.

1.

2. Use of the DNA polymerase III mutant of claim 1 in increasing the mutation frequency of the genome of a host bacterium, wherein the host bacterium is a recombinant Escherichia coli producing tryptophan, and the Escherichia coli is one of W3110, DH5a, and BL21.

3. Use according to claim 2, characterized in that, The method comprises the following steps: (1) cloning a wild-type DNA polymerase III gene of Escherichia coli to obtain a 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, and using a mutant primer to amplify the mutant dnaQ gene by PCR to obtain a second expression vector containing the DNA polymerase III mutant gene after one-step cloning; (3) transforming the second expression vector into a host bacterium to express the DNA polymerase III mutant.

4. Use according to claim 3, characterized in that, In step (1), the cloning method of the dnaQ gene comprises: using the genome of Escherichia coli as a template, and using the nucleic acid sequences shown as SEQ ID No. 3-4 as primers to amplify the dnaQ gene by PCR; and the method of inserting the dnaQ gene into the vector comprises: double enzyme digestion of a plasmid to obtain a linearized vector fragment, and connecting the dnaQ gene with the linearized vector fragment to obtain a circular first expression vector.

5. Use according to claim 3, characterized in that, In step (1), the vector is pBad24.

6. Use according to claim 3, characterized in that, In step (2), the method of amplifying by PCR using a mutant primer comprises: using the first primer pair shown as SEQ ID No. 5-6 to amplify the first expression vector to obtain a third expression vector, and using the second primer pair shown as SEQ ID No. 7-8 to amplify the third expression vector to obtain the second expression vector; or using the first primer pair shown as SEQ ID No. 7-8 to amplify the first expression vector to obtain a third expression vector, and using the second primer pair shown as SEQ ID No. 5-6 to amplify the third expression vector to obtain the second expression vector.

7. Use of the DNA polymerase III mutant of claim 1 in an adaptive evolution strain.

8. A plasmid containing the gene of the DNA polymerase III mutant of claim 1. ​

Images