DNA modification and transfer integrated escherichia coli engineering strain and application thereof

Abstract

The invention discloses a DNA (Deoxyribose Nucleic Acid) modification and transfer integrated escherichia coli engineering strain and application thereof. Three engineering strains of E.coli GB06-DLP12Tra-alpha beta gamma, GB05-recETTra-alpha beta gamma and DH5G-GTra-alpha beta gamma are constructed by integrating components with conjugational transfer and recombination functions, namely, Tra-red alpha beta gamma onto genomes of different Escherichia coli, and the E.coli GB06-DLP12Tra-alpha beta gamma has the best stability on repetitive sequences, so that plasmids containing 500bp direct repetitive sequences can stably exist, and the expression quantity of the E.coli GB06-DLP12Tra-alpha beta gamma is greatly increased. The possibility of unexpected intramolecular recombination of large plasmids in genetic manipulation is reduced as much as possible; when the E.coli GB05-recETTra-alpha beta gamma is used as a donor, the conjugational transfer efficiency is the highest, and is higher than that of an existing donor E.coli ET12567 expressed by a plasmid pUZ8002; the E.coli GB05-recETtra-alpha beta gamma and the DH5G-Gtra-alpha beta gamma have high electrotransfection efficiency on the plasmids, and the plasmids can be modified under the induction of rhamnose. The bifunctional engineering bacteria E.coli GB05-recETtrar-alpha beta gamma and DH5G-Gtrar-alpha beta gamma can be used for mining a natural product synthesis gene cluster, and the cloned gene cluster is directly transferred to a proper streptomyces host for heterologous expression after being modified.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to an Escherichia coli engineering strain integrating DNA modification and transfer and application thereof. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Conjugative transfer is widely present between bacteria, between bacteria and yeast, and between bacteria and mammalian cells. This process is initiated by the conjugative transfer element tra. Strains containing the conjugative transfer element tra can be used as donors to drive the transfer of plasmids containing oriT to other recipients. Due to its simple and convenient operation, conjugative transfer is the most commonly used method to transfer DNA from Escherichia coli to Streptomyces. Donors that mediate DNA transfer to Streptomyces include E. coli S17-1, in which the element tra is integrated into the chromosome, and E. coli ET12567, in which tra exists in the form of plasmid pUZ8002.

[0004] The target plasmid needs to contain oriT site in order to be recognized by Tra protein and transferred to other receptors. However, E. coli S17-1 and E. coli ET12567 / pUZ8002 do not have recombination function. Therefore, the target plasmid needs to be inserted into the oriT site in E. coli containing recombination system, such as E. coli GB05-red, and then electroporated into the receptor E. coli S17-1 or E. coli ET12567 / pUZ8002 for DNA transfer. If the plasmid contains more repetitive sequences and E. coli has poor stability to repetitive sequences, undesirable intramolecular recombination may occur during electroporation or recombination. The DNA modification and transfer process requires two strains of E. coli, two-step electroporation and two identifications of the plasmid. The process is repetitive, cumbersome and time-consuming.

[0005] When using E. coli S17-1 as the donor for transferring DNA, it was found that the target plasmid would be methylated by the methyltransferases (Dam and Dcm) of E. coli itself. After being transferred into Streptomyces with a strong methylation restriction system, it would be cleaved, resulting in low conjugation transfer efficiency. When using the methylation-deficient strain E. coli ET12567 / pUZ8002 as the donor, methylation of the plasmid in E. coli was avoided, thus improving the conjugation transfer efficiency. However, the electrotransformation efficiency of this donor was low. Since the element tra in this donor existed in the form of a plasmid, it would interfere with the identification of the target plasmid, and kanamycin was always required to maintain the existence of the plasmid pUZ8002. In addition, E. coli itself contained resistance genes for chloramphenicol and tetracycline, which led to the production of multiple resistances by E. coli ET12567 / pUZ8002, restricting its application. Therefore, it was necessary to develop an E. coli engineering strain that could stably maintain repetitive sequences and integrate DNA modification and transfer. There was no report in the relevant literature on carrying the Red recombination system and the tra conjugation transfer system in the same E. coli strain. Summary of the Invention

[0006] Aiming at the deficiencies existing in the prior art, the present invention provides an E. coli engineering strain integrating DNA modification and transfer and its application.

[0007] To achieve the above object, the present invention is realized through the following technical solutions:

[0008] In the first aspect, the present invention provides an E. coli engineering strain integrating DNA modification and transfer. The methyltransferase genes dcm and dam of the starting strain E. coli GB2005 are inactivated, and the bifunctional element tra-redαβγ is integrated into the position of the chromosomal gene recET of E. coli, thus obtaining the strain, named GB05-recETtra-αβγ. This engineering bacterium can not only perform DNA modification but also DNA transfer, and has the highest conjugation transfer efficiency among the four engineering bacteria.

[0009] In some embodiments, the application of GB05-recETtra-αβγ in DNA modification or DNA transfer.

[0010] In a second aspect, the present invention provides an integrated E. coli engineering strain for DNA modification and transfer. The methyltransferase genes dcm and dam of the starting strain E. coli DH5G are inactivated, the A at the 259th position of the DNA helicase gyrA* gene is changed to G, and the bifunctional element tra-redαβγ is integrated into the position of the E. coli chromosomal gene pspG, thus obtaining the strain, named E. coli DH5G-Gtra-αβγ. This engineering bacterium can perform both DNA recombination experiments and DNA transfer, but the conjugation transfer efficiency is relatively low.

[0011] In some embodiments, the application of E. coli DH5G-Gtra-αβγ in DNA modification or DNA transfer.

[0012] In a third aspect, the present invention provides an integrated E. coli engineering strain for DNA modification and transfer. Using E. coli GB2006 as the starting strain, the methyltransferase genes dcm and dam of the starting strain E. coli DH5G are inactivated, the A at the 259th position of the DNA helicase gyrA* gene is changed to G, and the bifunctional element tra-redαβγ is integrated into the position of the E. coli chromosomal gene ybcW, thus obtaining the strain, named E. coli GB06-DLP12tra-αβγ. This strain has the lowest conjugation transfer efficiency among the four engineering bacteria but is the most stable for plasmids containing repetitive sequences.

[0013] In some embodiments, the application of E. coli GB06-DLP12tra-αβγ in DNA modification or DNA transfer.

[0014] In a fourth aspect, the present invention provides an efficient E. coli engineering strain capable of DNA transfer with Streptomyces. Using E. coli S17-1 as the starting strain, all Mu genes on the chromosome are knocked out, and the methyltransferase genes dcm and dam are inactivated, thus obtaining the strain, named E. coli S17-1ΔMu. Since E. coli S17-1 itself already has the conjugation transfer element tra, only the methyltransferase genes dcm and dam are inactivated, and the conjugation transfer efficiency between E. coli and Streptomyces containing a methylation restriction system is improved.

[0015] In some embodiments, the application of E. coli S17-1ΔMu in DNA transfer.

[0016] In some embodiments, the method of inactivating dcm is to mutate the G at the 135th position in the dcm coding sequence to A. As a result, the codon TGG is mutated to the stop codon TGA, prematurely terminating gene transcription and thus inactivating the dcm gene.

[0017] In some embodiments, the way to inactivate dam is to knockout the coding region of the dam gene.

[0018] Inactivate dcm and dam to avoid the cleavage of foreign DNA by the methylation restriction system in Streptomyces and improve the efficiency of conjugation transfer.

[0019] The beneficial effects obtained by one or more of the above embodiments of the present invention are as follows:

[0020] The modification of gyrA, dcm, recA and dam genes on the chromosome of the Escherichia coli engineering strain is achieved by combining the Red recombination technology with a counter-selection marker to construct an engineering bacterium without a resistance marker, which is beneficial to the selection of resistance genes when subsequently modifying the target plasmid. Secondly, the redαβγ recombination system and the tra conjugation transfer system are integrated into the chromosome, which is beneficial to the stable inheritance of the system in Escherichia coli and reduces the interference with the identification of the target plasmid. Finally, the stability of the constructed strains to repetitive sequences is better than that of the commonly used conjugation transfer system E. coli ET12567 / pUZ8002. The establishment of the DNA modification and transfer integrated Escherichia coli engineering strain reduces the experimental steps and shortens the experimental time. This engineering bacterium is particularly suitable for the modification and transfer of natural product synthesis gene clusters.

[0021] The present invention provides four Escherichia coli engineering strains E. coli GB05-recETtra-αβγ, DH5G-Gtra-αβγ, GB06-DLP12tra-αβγ, and S17-1ΔMu that express the conjugation transfer element tra on the chromosome. Among them, the conjugation transfer efficiency of the strains E. coli GB05-recETtra-αβγ and S17-1Δmu is higher than that of the currently commonly used system E. coli ET12567 / pUZ8002; E. coli GB05-recETtra-αβγ and DH5G-Gtra-αβγ are engineering bacteria with high electroporation plasmid efficiency and capable of DNA modification and transfer; E. coli GB06-DLP12tra-αβγ is the engineering bacterium with the best stability to repetitive sequences.

[0022] The engineering bacteria E. coli GB05-recETtra-αβγ and E. coli DH5G-Gtra-αβγ can not only carry out DNA recombination experiments but also DNA transfer. Therefore, these two strains can be used for the modification and transfer of large natural product synthesis gene clusters.

[0023] E. coli GB06-DLP12tra-αβγ has excellent stability to plasmids containing repetitive sequences and can be used to transfer large natural product synthesis gene clusters with more repetitive sequences. Brief Description of the Drawings

[0024] The attached drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0025] Figure 1 : Schematic diagram of the internal recombination of plasmid pBAC-cm-ampF-kan-ampR-repea.

[0026] Figure 2 : Internal molecular recombination efficiency and electroporation efficiency of four candidate strains (E. coli GB2005, GB2006, DH5G, and S17-1). (a) Internal molecular recombination efficiency of four candidate strains. (b) Electroporation efficiency of four candidate strains.

[0027] Figure 3 : Process of modifying gyrA, dcm, and dam genes on the E. coli chromosome.

[0028] Figure 4 : Integration process of the bifunctional system tra-redαβγ.

[0029] Figure 5 : Functional evaluation results of four E. coli engineered strains (E. coli GB05-recETtra-αβγ, DH5G-Gtra-αβγ, GB06-DLP12tra-αβγ, and S17-1Δmu). (a) Stability to repetitive sequences; (b) Conjugation transfer efficiency; (c) Electroporation efficiency; (d) Recombination efficiency. Detailed Description of the Invention

[0030] It should be noted that the following detailed description is illustrative and aims to provide a further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0031] The present invention will be further described below in conjunction with embodiments.

[0032] All strains involved in the following examples, E. coli GB2005 (Method Enzymol. 2010, 477: 125-144), GB2006 (Microb Cell Fact. 2020, 19(1): 230), DH5G (Nucleic Acids Res. 2016, 44: 5365-5377), and S17-1 (Bio / Technology 1983, 1: 784-791), are all from the research group of Youming Zhang at Shandong University. The conjugation transfer element tra is from the plasmid pRK2013 (Proc Natl Acad Sci U S A. 1979, 76(4): 1648-1652);

[0033] The Redαβγ recombination system is from pSC101-P Rha -αβγA-P BAD -ccdA-tet (Nucleic Acids Res. 2013, 42(5): 37).

[0034] The electrotransformation method of Escherichia coli and the conjugation transfer operation between Escherichia coli and Streptomyces are conventional methods. In the following examples, other reagents and consumables involved are domestic. Unless otherwise specified, the experimental methods and reagents in the examples are all conventional methods in this field and commercially available reagents, which can be obtained through commercial channels.

[0035] Example 1

[0036] For the electrotransformation of Escherichia coli involved below, all operations are carried out according to this method. Specific steps of electrotransformation: 1) Pick a single colony of Escherichia coli on the LB plate and transfer it to a 1.5 mL centrifuge tube containing 1 mL of antibiotic-free LB liquid medium. Incubate overnight at 37 °C and 950 rpm on a shaker. 2) Transfer 40 μL of the overnight culture to a 1.5 mL centrifuge tube containing 1.3 mL of fresh LB liquid medium. Incubate at 37 °C and 950 rpm until the OD 600 reaches about 0.8, and then prepare Escherichia coli competent cells. Centrifuge at 9000 rpm for 1 min at room temperature to collect the cells and discard the supernatant. Resuspend with 1 mL of ddH2O and centrifuge, repeating the operation 2 times. 3) Resuspend the cells in 30 μL of ddH2O, add an appropriate amount of plasmid, mix well, transfer to a 1 mm electroporation cuvette, and perform electroporation at 1350 V. After electroporation, add 1 mL of antibiotic-free LB medium to the electroporation cuvette, pipette repeatedly, and then transfer to a perforated 1.5 mL centrifuge tube for recovery. 4) After recovering at 37 °C for one hour, perform appropriate dilution and then spread on an LB plate containing the corresponding antibiotic. Incubate inverted in a 37 °C incubator until colonies grow.

[0037] The specific scheme for constructing the E. coli engineering strain is as follows:

[0038] (1) Screening of candidate E. coli strains. Since some DNA molecules contain a large number of repetitive sequences, unnecessary intramolecular and intermolecular recombination may occur during DNA cloning or modification, reducing accuracy; the electroporation efficiency of the engineered strain also directly affects the efficiency of DNA modification, and different E. coli as donors will affect the efficiency of conjugation transfer.

[0039] First, the stability of different E. coli to repetitive sequences was tested. The molecular internal recombination efficiency of the plasmid pBAC-cm-ampF-kan-ampR-repeat containing the repetitive sequence was used to evaluate the stability of E. coli to repetitive sequences. The sequence of the plasmid pBAC-cm-ampF-kan-ampR-repeat is shown in SEQ ID No.1. The plasmid contains a chloramphenicol resistance gene (cm) and a kanamycin resistance gene (kan) with a 500bp direct repetitive sequence upstream and downstream. If the repetitive sequence is recombined, the complete ampicillin resistance gene (such as Figure 1 ). The plasmid was electrotransformed into E. coli. If it could replicate stably, the E. coli would show resistance to cm and kan. If the plasmid could not replicate stably in E. coli, intramolecular recombination occurred, kan resistance disappeared, and E. coli would show resistance to cm and amp. 1μg pBAC-cm-ampF-kan-ampR-repeat was electrotransformed into E. coli according to the above electrotransformation method. After one hour of recovery, it was appropriately diluted and spread on plates containing cm antibiotics and amp antibiotics. The intramolecular recombination efficiency was expressed by dividing the number of colonies grown on the amp antibiotic plate by the number of colonies grown on the cm antibiotic plate.

[0040] Figure 2 This is the result of the stability of E. coli to DNA repetitive sequences and the efficiency of electroporation. Figure 2 a It can be seen that the internal recombination efficiency of plasmid pBAC-cm-ampF-kan-ampR-repeat in E. coli GB2006 and E. coli S17-1 is 0, indicating that these two strains have the highest stability to the repetitive sequence and no unwanted internal molecular recombination occurs in the plasmid; followed by E. coli DH5G and finally E. coli GB2005, but the stability to the repetitive sequence is higher than that of E. coli ET12567.

[0041] according to Figure 2It can be seen from b that 2 μg of plasmid pBAC-sal-phiC31-apra-oriT was electrotransformed into 5 strains of Escherichia coli by the same electrotransformation method, and the plates containing apramycin antibiotic were used for screening. The number of colonies grown on the plates was the electrotransformation efficiency of the plasmid. The results showed that except for E. coli GB2006, the electrotransformation efficiencies of the other 3 strains of Escherichia coli were higher than that of the control group (E. coli ET12567). Among them, the electrotransformation efficiencies of E. coli GB2005 and E. coli DH5G were 356 and 157 times that of the control, significantly higher than that of E. coli ET12567. According to the results of the stability of Escherichia coli to DNA repeat sequences and electrotransformation efficiency (as Figure 2 shown), E. coli GB2005, GB2006, DH5G and S17-1 were selected as candidate strains for modification.

[0042] (2) Through the above Red recombination engineering, combined with the reverse selection marker ccdB (CcdB, as a toxic protein, can attack the topoisomerase of Escherichia coli, and its presence causes the death of Escherichia coli), the gyrA, dcm, dam and recA genes on the chromosomes of 4 strains of Escherichia coli were modified. Since the recombination systems of strains E. coli GB2005, GB2006, DH5G and S17-1 were inefficient, the recombinant enzyme expression plasmid pSC101-P Rha -αβγA-P BAD -ccdA-tet (Nucleic Acids Res. 2013, 42(5): 37) was used to complete the modification of the genes on the chromosomes of Escherichia coli. Adding 10% rhamnose can induce the expression of the recombination system redαβγ on the plasmid, so that the fragment with a 50 bp homologous arm can be inserted into the appropriate position on the chromosome; adding 10% arabinose can induce the expression of ccdA on the plasmid. CcdA is the antidote of the toxic protein CcdB, which facilitates the use of the reverse selection marker ccdB. After the modification, the recombinant enzyme expression plasmid can be lost by increasing the culture temperature (37°C).

[0043] The Red recombination engineering, the specific operation steps are as follows: 1) Pick a single colony of Escherichia coli on the LB plate and transfer it to a 1.5 mL centrifuge tube containing 1 mL of LB liquid medium. Incubate overnight at 37°C, 950 rpm (if the strain contains a plasmid with a temperature-sensitive replicon pSC101, culture at 30°C). 2) Take 40 μL of the culture and transfer it to a 1.5 mL centrifuge tube containing 1.2 mL of LB liquid medium. Incubate at 30°C, 950 rpm, and culture OD 600After about 0 to 0.8, add 10% rhamnose to induce the recombination system redαβγ (if ccdA needs to be induced, add 10% arabinose), and continue culturing at 37°C and 950 rpm for 40 min. 3) Pre-cool the centrifuge in advance. Under the condition of 4°C, centrifuge the bacterial cells at 9000 rpm for 1 min, discard the supernatant, and collect the bacterial cells. Add 1 mL of pre-cooled ddH2O, mix well, centrifuge at 9000 rpm for 1 min, discard the supernatant, and collect the bacterial cells. Repeat 2 times, and finally suspend with 30 μL of ddH2O. 4) Add 600 ng of the DNA fragment, mix well, transfer it to a pre-cooled 1 mm electroporation cuvette, and perform electroporation at 1350 V. After electroporation, add 1 mL of LB medium, pipette repeatedly, and then transfer it to a punctured 1.5 mL centrifuge tube for recovery. 5) After 1 hour of recovery, spread it on an LB plate containing the corresponding antibiotic, and culture at 37°C until colonies grow. Pick a single colony, verify by colony PCR or perform restriction enzyme digestion analysis with a suitable restriction enzyme, and pick the correct recombinant.

[0044] First, point mutations of gyrA in E. coli GB2006 and DH5G: Nalidixic acid is commonly used to kill E. coli after conjugation transfer between E. coli and Streptomyces. The gyrA* genes in E. coli GB2006 and DH5G have mutated and become resistant to nalidixic acid. To restore gyrA* to the wild-type gyrA and regain their sensitivity to nalidixic acid, the A at position 259 in the ORF region of the original gyrA* needs to be changed to G. The point mutation of the gyrA* gene was completed through three-step Red recombination engineering. The primer tables for all experiments are listed in Table 1. The specific operation steps are as follows: (1) Using kan-gyrA-1 and kan-2 as primers and pBR322-amp-ccdB-kan-rpsL as the template, PCR amplified the fragment kan; using gyrA-1 and kan-gyrA-2 as primers and the genome of E. coli GB2005 as the template, PCR amplified the fragment gyrA. For tandem PCR amplification, using kan-gyrA-1 and kan-gyrA-2 as primers and the fragments kan and gyrA as templates, PCR amplified and recovered the fragment kan-gyrA by agarose gel electrophoresis. (2) According to the method of Red recombination engineering, the recovered fragment kan-gyrA was electrotransformed into E. coli GB2006 and E. coli DH5G for recombination. Recombinants E. coli GB2006-kan-gyrA and E. coli DH5G-kan-gyrA were screened using plates containing kanamycin antibiotic and verified by colony PCR to complete the first round of Red recombination. (3) Using gyrA-amp-ccdb-1 and gyrA-amp-ccdb-2 as primers and pBR322-amp-ccdB-kan-rpsL as the template, PCR amplified the fragment amp-ccdB and recovered the fragment by agarose gel electrophoresis. According to the method of Red recombination engineering, after adding 10% rhamnose and arabinose and inducing for 40 min, the DNA fragment amp-ccdB was recombined into E. coli GB2006-kan-gyrA and E. coli DH5G-kan-gyrA. Recombinants E. coli GB2006-gyrA-amp-ccdB and E. coli DH5G-gyrA-amp-ccdB were screened using plates containing arabinose and ampicillin antibiotic and verified by colony PCR (subsequently, the culture medium needs to contain L-arabinose) to complete the second round of Red recombination. (4) According to the method of Red recombination engineering, the synthesized 99-bp single-stranded DNA fragments gyrA-99-1 and gyrA-99-2 were transferred into E. coli GB2006-gyrA-amp-ccdB and E. coli DH5G-gyrA-amp-ccdB for recombination.After resuscitation, recombinants E. coli GB2006-gyrA and E. coli DH5G-gyrA were screened on plates without any antibiotics and without L-arabinose, and colony PCR and sequencing verification were performed to complete the third round of Red recombination.

[0045] Secondly, point mutations were introduced into the methyltransferase gene dcm of E. coli GB2006-gyrA, DH5G-gyrA, GB2005, and S17-1. The G at position 135 in the coding sequence of dcm was mutated to A, resulting in the codon TGG being mutated to the stop codon TGA, which prematurely terminated gene transcription and inactivated the dcm gene. The primers involved in this process are listed in Table 1. The specific experimental procedure is as follows: First, using dcm-ccdB-1 and dcm-ccdB-2 as primers and pBR322-amp-ccdB-kan-rpsL as the template, the fragment amp-ccdB was amplified by PCR and recovered by agarose gel electrophoresis. According to the method of Red recombination engineering, after adding 10% rhamnose and arabinose and inducing for 40 min, the fragment amp-ccdB was recombined into the genomes of E. coli GB2006-gyrA, DH5G-gyrA, GB2005, and S17-1. Recombinants E. coli GB2006-gyrA-Δdcm-amp-ccdB, DH5G-gyrA-Δdcm-amp-ccdB, GB2005-Δdcm-amp-ccdB, and S17-1-Δdcm-amp-ccdB were screened on plates containing arabinose and ampicillin antibiotics, and verified by colony PCR (subsequently, the culture medium needs to contain L-arabinose) to complete the first round of Red recombination. Then, according to the method of Red recombination engineering, the synthesized 99-bp single-stranded DNA fragments dcm-99-1 and dcm-99-2 were transferred into E. coli GB2006-gyrA-Δdcm-amp-ccdB, DH5G-gyrA-Δdcm-amp-ccdB, GB2005-Δdcm-amp-ccdB, and S17-1-Δdcm-amp-ccdB for recombination. After resuscitation, screening was performed on plates without any antibiotics and without arabinose, and colony PCR and sequencing verification were carried out to complete the second round of Red recombination. Finally, the G at position 135 in the DNA sequence of dcm on the chromosome was mutated to A, and four strains E. coli DH5G-gyrA-Δdcm, GB06-gyrA-Δdcm, GB05-Δdcm, and S17-1-Δdcm were obtained.

[0046] Since the recA genes of E. coli DH5G-gyrA-Δdcm, GB06-gyrA-Δdcm, GB05-Δdcm, and S17-1-Δdcm are themselves mutated, and the double mutation of dam and recA genes is lethal to E. coli. Before knocking out dam, site-directed mutagenesis of recA in E. coli was performed by two-step Red recombination engineering. The method was similar to the site-directed mutagenesis of dcm. The primers used in the first round of recombination were recA-ccdB-1 and recA-ccdB-2. Using pBR322-amp-ccdB-kan-rpsL as a template, the DNA fragment amp-ccdB was amplified by PCR for recombination. The DNA fragment in the second round of Red recombination used recA-1 and recA-2 as primers, and the genome of E. coli ET12567 as a template. The fragment was amplified by PCR and recovered by agarose gel for recombination. Finally, four strains E. coli DH5G-gyrA-Δdcm-recA, GB06-gyrA-Δdcm-recA, GB05-Δdcm-recA, and S17-1-Δdcm-recA were obtained.

[0047] Finally, the entire coding region (0.8 kb) of the dam gene in E. coli DH5G-gyrA-Δdcm-recA, GB06-gyrA-Δdcm-recA, GB05-Δdcm-recA, and S17-1-Δdcm-recA was knocked out, resulting in the inactivation of the dam gene to avoid the cleavage of foreign DNA by the methylation restriction system in Streptomyces and improve the efficiency of conjugation transfer. The knockout method was similar to that of the dcm gene. Through two rounds of Red recombination engineering, the primers required for preparing the DNA fragment in the first round of Red recombination engineering were dam-ccdB-1 and dam-ccdB-2, and the second round was the synthesized 99-bp single-stranded DNA fragments dam-99-1 and dam-99-2. Four methylation-deficient strains E. coli DH5G-gyrA-Δdcm-recA-Δdam, GB06-gyrA-Δdcm-recA-Δdam, GB05-Δdcm-recA-Δdam, and S17-1-Δdcm-recA-Δdam were obtained.

[0048] The entire genome of Mu phage is contained on the chromosome of E. coli S17-1-Δdcm-recA-Δdam, and it can transfer some DNA on the chromosome to the recipient at a certain frequency. Therefore, we deleted the entire genome (39 kb) of Mu phage to prevent such accidental events. The Mu gene was knocked out using the same two rounds of Red recombination engineering. The primers required for preparing the recombinant DNA fragments in the first round of Red recombination engineering were Mu-ccdB-1 and Mu-ccdB-2, and in the second round, they were the synthetic 99-bp single-stranded DNA fragments Mu-99-1 and Mu-99-2. The final engineered bacterium was obtained and named E. coli S17-1-ΔMu.

[0049] Table 1 Primers for DNA fragments required for gyrA, dcm, recA, dam, and Mu recombination

[0050]

[0051]

[0052] Note: Lowercase letters are homologous arms.

[0053] (3) Integration of the bifunctional system tra-redαβγ on the E. coli chromosome (as Figure 4 shown):

[0054] Construction of the plasmid carrying the bifunctional element tra-redαβγ: Seven DNA fragments, p15A-amp-ccdB, intλ, attP1, zeo, tra, redαβγ, and attP2, were prepared according to Table 2. Table 3 shows the primers required for the prepared DNA fragments, and they were recombined into the plasmid p15A-amp-ccdB-intλ-attP1-zeo-tra-redαβγ-attP2 carrying the bifunctional element tra-redαβγ through the ExoCET technique (Nucleic Acids Res. 2018, 46(5): e28) (as Figure 4 shown). Intλ is the integrase; attP1 and attP2 are the recognition sites of the integrase Intλ, which can recombine with attB1 and attB2 on the chromosome; CcdB serves as a counter-selection marker to ensure that the element tra-redαβγ is integrated into the chromosome rather than existing as a free plasmid; amp and zeo are resistance marker genes.

[0055] Table 2 DNA fragments required for constructing the plasmid carrying the bifunctional element tra-redαβγ

[0056]

[0057] Table 3 Primers required for constructing plasmids carrying the bifunctional element tra-redαβγ

[0058]

[0059] Note: Lowercase letters are homologous arms.

[0060] Integration of the bifunctional element tra-redαβγ: First, attB1-cm-attB2 was inserted into the chromosome of Escherichia coli. The primers are shown in Table 4. Using pRK-lox71-cm-lox66 as a template, three DNA fragments HA-attB1-cm-attB2-HA (HA represents the homologous arms of the pspG, ybcW, or recET locus) with 50-bp homologous arms were amplified by PCR and purified and recovered. Through the above-mentioned Red recombination engineering, 200 ng of the fragment HA-attB1-cm-attB2-HA was electrotransformed into methylation-deficient E. coli DH5G-gyrA-Δdcm-recA-Δdam, GB06-gyrA-Δdcm-recA-Δdam, and GB05-Δdcm-recA-Δdam for recombination. Recombinants E. coli DH5G-pspG-attB, GB06-ybcW-attB, and GB05-recET-attB were screened using cm-resistant plates. Then, the plasmid p15A-amp-ccdB-intλ-attP1-zeo-tra-redαβγ-attP2 carrying the bifunctional element tra-redαβγ was electrotransformed into E. coli DH5G-pspG-attB, GB06-ybcW-attB, and GB05-recET-attB according to the above electrotransformation method. After recovery for 2.5 h, it was spread on plates containing zeo antibiotic. Through Intλ-mediated site-specific recombination, attP1 and attP2 on the plasmid recombined with attB1 and attB2 on the chromosome, and the bifunctional element tra-redαβγ was exchanged with the cm resistance gene on the chromosome and integrated into the chromosome of Escherichia coli. Finally, the engineered E. coli strains E. coli DH5G-Gtra-αβγ, GB06-DLP12tra-αβγ, and GB05-recETtra-αβγ were obtained.

[0061] Table 4 Primers required for the fragment HA-attB1-cm-attB2-HA

[0062]

[0063] Note: Lowercase letters are homologous arms, and the underlined lowercase letters are the DNA sequences of attB1 and attB2.

[0064] Through the above scheme, a total of 4 engineered Escherichia coli strains were constructed, named E.coli GB05-recETtra-αβγ, DH5G-Gtra-αβγ, GB06-DLP12tra-αβγ and S17-1ΔMu, and their genotypes are shown in Table 5.

[0065] Table 5 Genotypes of the four engineered bacteria

[0066]

[0067] Functional characterization was carried out on the stability of the repetitive sequences, conjugation transfer efficiency, electroporation efficiency and recombination efficiency of the four engineered strains, and the results are as follows ( Figure 5 ):

[0068] (1) The stability of the strains was compared by the internal recombination rate of the plasmid pBAC-cm-ampF-kan-ampR-repeat containing the repetitive sequence. The lower the internal recombination rate, the more stable the strain is to the repetitive sequence. Taking E.coli ET12567 as the control, the stability of the four engineered bacteria to the repetitive sequence is as Figure 5 shown in a: The internal recombination rate of the plasmid in E.coli GB06-DLP12tra-αβγ is 0, and no internal recombination of the plasmid occurs, indicating that this engineered bacterium has significant stability to the repetitive sequence; followed by E.coliGB05-recETtra-αβγ, S17-1Δmu and DH5G-Gtra-αβγ, which are 25%, 31% and 46% of E.coli ET12567 respectively. The stability of the four engineered bacteria to the plasmid is better than that of E.coli ET12567.

[0069] (2) For the conjugation transfer efficiency test, the specific experimental operations are as follows: 1) Pick the Escherichia coli donor bacteria containing the plasmid on the LB plate and inoculate them into a 2 mL centrifuge tube containing 1.8 mL of LB liquid medium with the corresponding antibiotic. Incubate overnight at 37 °C and 950 rpm on a shaker. 2) Transfer 1.5 mL of the overnight culture to 15 mL of LB liquid medium with the corresponding antibiotic and culture at 37 °C until OD 600After reaching about 0.6, centrifuge at 9000 rpm, collect the bacterial cells and discard the supernatant. Suspend the cells in 10 mL of ddH2O and centrifuge again. Repeat this process twice. Then resuspend the cells in 5 mL of ddH2O to serve as the donor. 3) Use an inoculation loop to scrape the spores of Streptomyces grown on the MS plate and transfer them into 800 μL of 2×YT medium. Vortex to mix well. Place the mixed spores in a preheated water bath at 50 °C for heat shock for 10 min, then immediately take them out and cool them in ice water for 5 min to serve as the recipient. 4) Take 1 mL of the donor and 100 μL of the recipient and place them in a 1.5 mL centrifuge tube. Vortex to mix well and spread them on a non-resistant MS plate (containing 10 mmol / L MgCl2). 5) Incubate the plate upside down in a 30 °C incubator for 17 h. Then evenly cover the integrated MS plate with 800 μL of ddH2O containing nalidixic acid (NA, used for the separation of the donor and the recipient) and the required antibiotics (for the screening of the conjugants). Continue to incubate at 30 °C. After single colonies grow, perform colony PCR verification. The conjugation transfer efficiency is calculated by dividing the number of colonies of the conjugants by the number of colonies of the recipient bacteria.

[0070] Using four strains of Escherichia coli engineering bacteria containing plasmid pBAC-sal-phiC31-apra-oriT as the donors and Streptomyces coelicolor A3(2) as the recipient bacteria, conjugation transfer was carried out to compare the ability of the four strains of engineering bacteria to transfer plasmid pBAC-sal-phiC31-apra-oriT. The results of the conjugation transfer efficiency are as Figure 5 shown in Fig. b: Taking E. coli ET12567 / pUZ8002 as the control, the conjugation transfer efficiencies of E. coli GB05-recETtra-αβγ and S17-1Δmu are 2 times and 1.7 times that of the control respectively. However, the transfer efficiencies of DH5G-Gtra-αβγ and GB06-DLP12tra-αβγ are lower than that of the control group, being 6.6% and 3.5% of the control group respectively. Although the transfer efficiency is low, in the engineering bacteria, the tra element is expressed on the chromosome. Compared with E. coli ET12567 in which pUZ8002 only exists under the external pressure of kanamycin, the tra inheritance in the engineering bacteria is more stable and does not interfere with the identification of the target plasmid.

[0071] (3) According to the above electroporation method, 2 μg of plasmid pBAC-sal-phiC31-apra-oriT (116 kb) was electroporated into the engineering bacteria, and screened with a plate containing apra antibiotic. The electrotransformation efficiency of the engineering bacteria for the plasmid was compared. The number of colonies growing on the plate represents the electrotransformation efficiency. Taking E. coli ET12567 / pUZ8002 as the control, the results are as Figure 5As shown in c: The transformation efficiencies of E. coli GB05-recETtra-αβγ, DH5G-Gtra-αβγ, and S17-1ΔMu are 41.9, 17.5, and 2.3 times that of E. coli ET12567 respectively, and E. coli GB06-DLP12tra-αβγ is 11% of the control group.

[0072] (4) Verification of the recombination efficiency of the engineered E. coli strains. Since the rhamnose-inducible promoter P Rha has a high expression intensity and good stringency in E. coli, therefore, P Rha is used to control the expression of the recombinase gene redαβγ. First, the plasmid pBAC-sal-phiC31-apra-oriT was electrotransformed into E. coli GB05-recETtra-αβγ, DH5G-Gtra-αβγ, and GB06-DLP12tra-αβγ. According to the experimental steps of the above Red recombination, the PCR fragment amp-PhiBT1 with 50 bp homologous arms was electrotransformed into the engineered bacteria containing the plasmid. After recovery for one hour, it was spread on the LB plate containing amp. The fragment amp-PhiBT1 recombined with the plasmid pBAC-sal-phiC31-apra-oriT, and the recombination efficiency could be represented by the number of colonies on the LB plate containing amp antibiotic. The recombination results showed ( Figure 5 d), both E. coli GB05-recETtra-αβγ (45) and E. coli DH5G-Gtra-αβγ (80) could complete the modification of DNA, while no recombination occurred in E. coli GB06-DLP12tra-αβγ and no colonies grew. Therefore, under the current experimental conditions, E. coli GB06-DLP12tra-αβγ could not complete the modification of DNA.

[0073] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An engineered Escherichia coli strain with integrated DNA modification and transfer, characterized in that: The methyltransferase genes dcm and dam of the starting strain E. coli GB2005 were inactivated, and the bifunctional element tra-redαβγ was integrated into the position of the E. coli chromosomal gene recET, named GB05-recETtra-αβγ.

2. Use of GB05-recETtra-αβγ according to claim 1 in DNA modification or DNA transfer.

3. An engineered Escherichia coli strain with integrated DNA modification and transfer, characterized in that: The methyltransferase genes dcm and dam of the starting strain E. coli DH5G were inactivated, the A at position 259 of the DNA helicase gyrA* gene was changed to G, and the bifunctional element tra-redαβγ was integrated into the position of the E. coli chromosomal gene pspG, and the result was named E. coli DH5G-Gtra-αβγ.

4. Use of the E. coli DH5G-Gtra-αβγ according to claim 3 in DNA modification or DNA transfer.

5. An engineered Escherichia coli strain with integrated DNA modification and transfer, characterized in that: Using E. coli GB2006 as the starting strain, the methyltransferase genes dcm and dam of the starting strain E. coli DH5G were inactivated, the A at position 259 of the DNA helicase gyrA* gene was changed to G, and the bifunctional element tra-redαβγ was integrated into the position of the E. coli chromosomal gene ybcW, and the obtained strain was named E. coli GB06-DLP12tra-αβγ.

6. Use of the E. coli GB06-DLP12tra-αβγ according to claim 5 in DNA transfer.

7. An engineered strain of Escherichia coli that is highly efficient in DNA transfer with Streptomyces, characterized in that: Using E. coli S17-1 as the starting strain, the Mu gene on the chromosome was completely knocked out, and the methyltransferase genes dcm and dam were inactivated to obtain E. coli S17-1ΔMu.

8. The engineered Escherichia coli strain according to claim 7, characterized in that: The method of inactivating dam is to knock out the coding region of the dam gene.

9. Use of the E. coli S17-1ΔMu according to claim 7 in DNA transfer.

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