An engineered bacterium and a construction method and application thereof

Abstract

The application provides an engineering bacterium and a construction method and application thereof. The first aspect of the application provides an engineering bacterium, which is Escherichia coli overexpressing a methylation enzyme for methylation modification of an exogenous gene; and the amino acid sequence of the methylation enzyme is shown in SEQ ID NO. 1 or SEQ ID NO. 3. The engineering bacterium provided by the application can perform methylation modification on an exogenous gene, improve the transformation efficiency of the exogenous gene in S. calvus, and provide an important basis for genetic engineering modification of S. calvus.

Description

TECHNICAL FIELD

[0001] The present application relates to an engineering bacteria and a construction method and application thereof, and relates to the technical field of genetic engineering. BACKGROUND

[0002] Foreign DNA transformation technology is the basis and core of the development of genetic engineering and DNA cloning technology, and is one of the most important operation methods in modern molecular biology. Common transformation methods include chemical transformation and electroporation, and generally have relatively high transformation efficiency for standard laboratory strains. However, for some strains screened in the environment or industrial strains, the transformation efficiency of foreign DNA is generally low.

[0003] For example, butenyl-spinosyns are a new type of macrolide natural product with high biological insecticidal activity, and are ideal green biological insecticides. The common production method is to use the secondary metabolism of soil actinomycete Saccharopolyspora pogona. However, the wild type Saccharopolyspora pogona has weak ability to synthesize butenyl-spinosyns and low yield, and it is necessary to use genetic engineering technology to transform Saccharopolyspora pogona to obtain an engineering strain with high yield of butenyl-spinosyns. However, Saccharopolyspora pogona has a strong restriction modification system, and when foreign genes enter the cell, they will be quickly degraded, making it very difficult to genetically manipulate Saccharopolyspora pogona, which limits the construction of high-yield butenyl-spinosyns engineering strains.

[0004] The restriction modification system generally includes a restriction enzyme and a modification enzyme. The restriction enzyme is a kind of endonuclease which can cut foreign DNA, and the modification enzyme can methylate the self DNA. In order to overcome the restriction modification system of Saccharopolyspora pogona, the modified foreign genes can be methylated to prevent them from being cut and degraded by the restriction modification system of Saccharopolyspora pogona, thereby improving the transformation efficiency of foreign genes. Therefore, how to methylate foreign genes to improve the transformation efficiency of foreign genes in Saccharopolyspora pogona has attracted continuous attention from those skilled in the art. SUMMARY

[0005] The present application provides an engineering bacteria which can overexpress a methylation enzyme as shown in SEQ ID NO. 1 or SEQ ID NO. 3, and can methylate foreign genes to improve the transformation efficiency of foreign genes in Saccharopolyspora pogona.

[0006] The present application also provides a construction method of the above engineering bacteria.

[0007] The application also provides a method for improving the transformation efficiency of an exogenous gene in S. pogona, which comprises introducing the exogenous gene into the above-mentioned engineering bacteria and performing methylation modification to improve the transformation efficiency of the exogenous gene in S. pogona.

[0008] The first aspect of the application provides an engineering bacteria, which is E. coli capable of overexpressing a methylation enzyme for methylation modification of an exogenous gene.

[0009] The amino acid sequence of the methylation enzyme is shown in SEQ ID NO. 1 or SEQ ID NO. 3.

[0010] The methylation enzyme provided by the application is derived from S. pogona (S. pogona ASAGF58). Through bioinformatics alignment analysis, it is found that there are three methylation enzymes on the genome of S. pogona, which are named methylation enzyme 04455, 28970 and 29090, respectively. The amino acid sequence of the methylation enzyme 04455 is shown in SEQ ID NO. 1, the amino acid sequence of the methylation enzyme 28970 is shown in SEQ ID NO. 3, both of which can perform methylation modification on an exogenous gene and improve the transformation efficiency of the exogenous gene in S. pogona. Specifically, the transformation efficiency of the methylation enzyme 28970 is 20.5 CFU / ug DNA, the transformation efficiency of the methylation enzyme 04455 is 57.5 CFU / ug DNA, and the amino acid sequence of the methylation enzyme 29090 is shown in SEQ ID NO. 5, which does not improve the transformation efficiency of the exogenous gene in S. pogona. Therefore, the construction of an engineering bacteria capable of overexpressing the methylation enzyme 04455 or 28970 can perform methylation modification on an exogenous gene and improve the transformation efficiency of the exogenous gene in S. pogona, which provides an important basis for subsequent genetic engineering of S. pogona.

[0011] In a preferred embodiment, based on the higher transformation efficiency of the methylation enzyme 04455 on an exogenous gene in S. pogona, an engineering bacteria capable of overexpressing the methylation enzyme 04455 is constructed, and the amino acid sequence of the methylation enzyme 04455 is shown in SEQ ID NO. 1.

[0012] Further, the E. coli is ET12567, which is a methylation-deficient strain. The introduced gene sequence will not be degraded by the methylation modification system of the strain, and it is a common strain for Streptomyces gene knockout or gene duplication. Those skilled in the art can purchase it according to conventional technical means in the art.

[0013] The second aspect of the application provides a construction method of the above-mentioned engineering bacteria, which comprises: integrating a gene encoding the methylation enzyme into the genome of E. coli to realize overexpression of the methylation enzyme and obtain the engineering bacteria.

[0014] The amino acid sequence of the methylase is shown in SEQ ID NO. 1 or SEQ ID NO. 3.

[0015] In one specific embodiment, the method comprises:

[0016] Step 1, connecting the gene encoding the methylase, the promoter J23119 and the ribosome binding site B0034 to construct the expression cassette of the methylase.

[0017] Specifically, the nucleotide sequence of the gene encoding the methylase 04455 is shown in SEQ ID NO. 2, and the nucleotide sequence of the gene encoding the methylase 28970 is shown in SEQ ID NO. 4 after codon optimization.

[0018] The expression of the gene encoding the methylase in E. coli requires the driving action of the promoter and the ribosome binding site. Specifically, the promoter used in the present application is the promoter J23119, and the ribosome binding site is B0034, which can initiate the expression of the downstream gene and has high strength. Specifically, the promoter J23119 and the ribosome binding site B0034 can be referred to in the literature (Systematic Analysis of Escherichia coli Isolates from Sheep and Cattle Suggests Adaption to the Rumen Niche), or can be obtained by querying the website http: / / parts.igem.org / Main_Page.

[0019] In addition, in order to screen the positive transformants, the construction of the expression cassette also needs to add a resistance sequence. Specifically, the resistance sequence used in the present application is the Kan resistance sequence. During the screening of the transformants, the E. coli after transformation is cultured in a culture medium containing spectinomycin, and the enzyme expressed by the Kan resistance sequence can degrade spectinomycin, so that the E. coli grows normally, thereby screening the positive transformants.

[0020] According to the conventional technical means in the art, Figure 1 The promoter J23119, the ribosome binding site B0034, the gene sequence encoding the methylase and the Kan resistance sequence are sequentially connected in the order to construct the expression cassette of the methylase.

[0021] Step 2, culturing the competent E. coli and introducing the expression cassette into the competent E. coli to screen the positive transformants to obtain the engineering bacteria.

[0022] The application realizes the introduction of the exogenous DNA molecule by culturing the competent cell. First, the E. coli is induced to be the competent cell. Specifically, the pTKRED plasmid is introduced into the E. coli by the method of electric shock transformation or chemical transformation, and is cultured under certain conditions. The single clone is picked and subjected to liquid overnight culture. Then, the IPTG is added for induction, so as to prepare the competent E. coli.

[0023] Secondly, the expression cassette prepared in step 1 is mixed with the competent E. coli, and the expression cassette with the target gene is introduced into the competent E. coli by electrode transformation. Finally, the E. coli is cultured, and the positive transformants are screened, so as to obtain the engineering bacteria.

[0024] The third aspect of the application provides the application of the above engineering bacteria in the methylation modification of the exogenous gene and the improvement of the transformation efficiency of the exogenous gene in the S. cichorii.

[0025] The above engineering bacteria provided by the application can perform the methylation modification on the exogenous gene, so as to obtain the exogenous gene after methylation modification. The exogenous gene after methylation modification can be introduced into the S. cichorii, and the transformation efficiency is improved.

[0026] The “exogenous gene” in the application can be any gene which needs to be transformed into the S. cichorii, and the application does not make any limitation on the exogenous gene.

[0027] The fourth aspect of the application provides a method for improving the transformation efficiency of the exogenous gene in the S. cichorii, which comprises the following steps.

[0028] The exogenous gene is introduced into the above engineering bacteria to perform the methylation modification, so as to obtain the exogenous gene after methylation modification.

[0029] The exogenous gene after methylation modification is introduced into the S. cichorii.

[0030] In a specific embodiment, the method comprises the following steps.

[0031] Step 1, the exogenous gene is introduced into the above engineering bacteria to perform the methylation modification, so as to obtain the exogenous gene after methylation modification.

[0032] Step 1.1, a recombinant vector comprising the exogenous gene is constructed, and the recombinant vector is introduced into the engineering bacteria to perform the methylation modification.

[0033] The exogenous gene which needs to be introduced into the S. cichorii is inserted into the plasmid, so as to construct the recombinant vector comprising the exogenous gene. The recombinant vector is introduced into the engineering bacteria by the electrode transformation method and other conventional technical means. The methylation modification is performed on the recombinant vector by the methylation enzyme expressed by the engineering bacteria.

[0034] Step 1.2, extract DNA of the engineering bacteria to obtain the methylation-modified recombinant vector;

[0035] After the bacteria grow up, the methylation-modified recombinant vector is extracted.

[0036] Step 2, the methylation-modified exogenous gene is introduced into the S. pogona.

[0037] The methylation-modified recombinant vector is introduced into the S. pogona by the method of protoplast transformation.

[0038] In conclusion, the engineering bacteria provided by the present application can overexpress the methylases 04455 and 28970, and the exogenous gene is subjected to methylation modification, thereby improving the transformation efficiency of the exogenous gene in the S. pogona, and providing an important foundation for the genetic engineering of the S. pogona. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 A is the construction principle diagram of the methylase 28970 expression cassette, B is the construction principle diagram of the methylase 04455 expression cassette, and C is the construction principle diagram of the methylase 29090 expression cassette;

[0040] Figure 2 It is a colony PCR verification diagram of ET12567::04455, ET12567::28970 and ET12567::29090;

[0041] Figure 3 It is a plate diagram of pSET-159-bpsA transforming S. pogona;

[0042] Figure 4 It is the transcription level of the genes encoding the methylases 04455 and 28970;

[0043] Figure 5 It is the SDS-PAGE analysis of the methylases 04455 and 28970;

[0044] Figure 6A It is the mass spectrum spectrum of the methylase 04455;

[0045] Figure 6B It is the mass spectrum spectrum of the methylase 28970. DETAILED DESCRIPTION

[0046] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0047] Construction of the engineered bacteria in Example 1

[0048] Step 1, cloning of the target fragment

[0049] The upstream homology arm was amplified with HA-F / R as primers and the ET12567 genome as a template; the downstream homology arm was amplified with HB-F / R as primers and the ET12567 genome as a template; the Kan resistance sequence was amplified with K-F / R as primers and the plasmid pKD3 as a template; the target sequence was obtained by amplifying the gene fragment (SEQ ID NO. 2) encoding methylase 04455 after codon optimization with 04455-F / R as primers; the expression cassette was obtained by assembling the above four fragments with HA-F / R as primers.

[0050] Similarly, the upstream homology arm was amplified with HA-F / R as primers and the ET12567 genome as a template; the downstream homology arm was amplified with HB-F / R as primers and the ET12567 genome as a template; the Kan resistance sequence was amplified with K-F / R as primers and the plasmid pKD3 as a template; the target sequence was obtained by amplifying the gene fragment (SEQ ID NO. 4) encoding methylase 28970 after codon optimization with 28970-F / R as primers; the expression cassette was obtained by assembling the above four fragments with HA-F1 and HB-R1 as primers.

[0051] Similarly, the upstream homology arm was amplified with HA-F / R as primers and the ET12567 genome as a template; the downstream homology arm was amplified with HB-F / R as primers and the ET12567 genome as a template; the Kan resistance sequence was amplified with K-F / R as primers and the plasmid pKD3 as a template; the target sequence was obtained by amplifying the gene fragment (SEQ ID NO. 6) encoding methylase 29090 after codon optimization with 29090-F / R as primers; the expression cassette was obtained by assembling the above four fragments with HA-F / R as primers.

[0052] The primer sequences are shown in Table 1.

[0053] Table 1 primer names and sequences used in Example 1

[0054]

[0055]

[0056] Step 2, the pTKRED plasmid was transformed into E. coli ET12567 by electric shock method, and was cultured overnight at 30°C on LB plates coated with spectinomycin. Single colonies were picked and cultured overnight in liquid. After the end of the culture, the inoculation was performed at a rate of 1%, and IPTG was added to induce at a final concentration of 1 mM. When the OD value reached about 0.8, the competent cells were prepared.

[0057] Step 3, the three expression cassettes constructed in step 1 were slowly added to the competent cells respectively, mixed well, and then subjected to electric shock transformation. Immediately after the electric shock transformation, 700 μL of LB medium was added, and the electrically transformed bacterial solution was transferred to a new sterile centrifuge tube. The solution was cultured at 37°C for 1 h, and then centrifuged at 5000 rpm for 2 min. The bacterial precipitate was collected and plated on LB solid medium containing the corresponding antibiotics, and cultured overnight at 37°C until single colonies were grown. The recombinants were picked and subjected to colony PCR using primers T-F / T-04455-R (T-28970-R / T-29090-R), respectively. The PCR program is shown in Table 2, and the verification results are shown in Figure 2 Table 3. The results show that the DNA band size is as expected, indicating that the expression cassette containing the target gene has been successfully integrated into the gene of E. coli ET12567. The constructed engineering strains are named ET12567::04455, ET12567::28970 and ET12567::29090, respectively.

[0058] Table 2 PCR program

[0059]

[0060]

[0061] Example 2 Introduction of exogenous genes

[0062] The plasmid pSET159-bpsA carries the gene encoding the synthesis of indigotin. After being transferred to the host, it appears blue on the plate, indicating that the plasmid has been successfully introduced. This is used to reflect the transformation efficiency of the exogenous gene in S. seton.

[0063] Step 1, the plasmid pSET159-bpsA was transformed into the engineering bacteria ET12567::04455, ET12567::28970 and ET12567::29090 by electric shock transformation to achieve methylation modification. After the colony grew, the pre-methylation plasmid pSET159-bpsA(m) was extracted by the plasmid extraction kit (Tiangen), and the extracted plasmid was uniformly quantified to 100 ng / μL. The plasmid pSET159-bpsA transformed into the ET12567 strain was used as a control group (Control).

[0064] Step 2, S. pogona protoplast transformation

[0065] (1) S. pogona was cultured in TSB medium, and glycine was added to a final concentration of 0.2%, and cultured at 4°C for 48h. After the culture was completed, the bacterial liquid was centrifuged at 5000rpm for 5min, and the mycelium was collected;

[0066] (2) The mycelium was washed twice with 10% sucrose solution, and the mycelium was resuspended with 10mL P buffer;

[0067] (3) The lysozyme solution was prepared with P buffer to a final concentration of 2mg / mL, and incubated at 30°C for about 30min. During the incubation, the centrifuge tube was gently inverted every 5min to promote the release of protoplasts;

[0068] (4) The bacterial liquid was filtered with a filter tube containing absorbent cotton, and the protoplasts were collected by centrifugation at 3000rpm for 5min;

[0069] (5) The protoplasts were plated on R5 medium to detect whether the protoplasts were contaminated and their activity;

[0070] (6) The protoplasts were suspended with 1mL P buffer, and immediately aliquoted into small portions (each portion added with 200μL protoplasts) and stored at -80°C or subjected to transformation.

[0071] (7) 50μL plasmid (pSET159-bpsA or methylated plasmid) and 50μL 60% PEG 4000 were added to 200μL prepared protoplast suspension, respectively, and mixed by gently tapping the tube wall with fingers. The suspension was plated on a dried R5 plate, and incubated at 30°C under regenerative culture temperature. When the regenerated protoplasts appeared as a mist on the plate (24h), an appropriate concentration of antibiotic aqueous solution was plated on the plate, and the culture was continued until colonies grew.

[0072] As Figure 3As shown in Table 3, compared with the control group, the number of colonies in the experimental groups 28970 and 04455 increased significantly, indicating that the transformation efficiency of the methylation-modified plasmid pSET159-bpsA in Streptosporangium flavidum was greatly improved. Specifically, the pSET159-bpsA plasmid was transformed into ET12567::28970, and the transformation efficiency was 20.5 CFU / μg DNA, which was 5.8 times higher than that of the control group; the pSET159-bpsA plasmid was transformed into ET12567::04455, and the transformation efficiency was 57.5 CFU / μg DNA, which was 16.4 times higher than that of the control group; and the pSET159-bpsA plasmid was transformed into ET12567::29090, and the transformation efficiency was lower than that of the control group.

[0073] Table 3 Transformation efficiency of plasmid pSET159-bpsA in Streptosporangium flavidum

[0074]

[0075] Example 4 Verification of transcription level of methylase 04455 and 28970

[0076] The RNA extraction method refers to the RNA extraction kit instruction, the RNA reverse transcription into cDNA operation method refers to the SYBR Green kit instruction, and the calculation method adopts the relative quantitative algorithm-ΔΔCt. According to the Ct of the standard product (normal) gene in the detection sample and the calibration sample, the Ct of the target gene (GOI) in the same two samples is adjusted, and the ΔΔCt value obtained can be used to determine the fold difference of expression. The specific formula is as follows:

[0077] Fold difference = 2 -ΔΔCt (1)

[0078] ΔΔC t = ΔC t样本 - ΔC t标准品 (2)

[0079] ΔC t样本 = C t GOI s – C t正常 s (3)

[0080] ΔC t校准品 = C t GOI c – C t正常 c (4)

[0081] Transcriptional level analysis of methylase 04455 and 28970 in S. pogona ASAGF58 was performed, and it was confirmed that both genes were transcribed. The engineered bacteria ET12567::04455 and ET12567::28970 were cultured and RNA was extracted, and the results are shown in Figure 4 Figure 6, the transcriptional level of methylase 04455 and 28970 in the engineered bacteria was much higher than that in S. pogona ASAGF58, specifically, the transcriptional level of methylase 28970 was increased by 40 times, and the transcriptional level of methylase 04455 was increased by 180 times.

[0082] Example 5 Protein level verification of methylase 04455 and 28970

[0083] In order to verify the expression of methylase in ET12567::04455 and ET12567::28970 at the protein level, mass spectrometry identification was performed, which specifically included the following steps:

[0084] (1) The fermentation broth of the engineered bacteria was centrifuged at 4°C, 5000 rpm for 10 min;

[0085] (2) The supernatant was removed, and a certain amount of PBS (pH 7.0) solution was used to resuspend the bacterial cells;

[0086] (3) 80 μL of the above sample was taken, 20 μL of 5×SDS loading buffer was added, and it was boiled for 10 min;

[0087] (4) Protein detection: 80 μL of whole cell sample was subjected to electrophoresis, and when the bromophenol blue indicator entered the separation gel, the voltage was 12 V / cm constant voltage. After the indicator was electrophoresed to the bottom of the separation gel 0.5 cm, the electrophoresis was stopped, and the results are shown in Figure 5 Figure 7.

[0088] The gel strip was sent to Huada Gene for protein mass spectrometry detection. The identification results of gel strip 04455 showed that the molecular weight was 53647.6 and the abundance was 71593.26. The identification results of gel strip 28970 showed that the molecular weight was 56658.73 and the abundance was 11470.93, indicating that methylase 04455 was detected in the engineered bacteria ET12567::04455, and the mass spectrometry spectrum is shown in Figure 6A Figure 8. Methylase 28970 was detected in the engineered bacteria ET12567::28970, and the mass spectrometry spectrum is shown in Figure 6B Figure 9, indicating that both modification enzymes were successfully expressed, and the alignment results of the two modification enzymes were consistent with the predicted results.

[0089] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. The application of an engineered bacterium in methylating a foreign gene and improving the transformation efficiency of the foreign gene in Polyspora sacchariformis; The engineered bacteria is *E. coli* capable of overexpressing methyltransferases, which are used to methylate foreign genes; the *E. coli* strain is a methylation-deficient strain. The amino acid sequence of the methyltransferase is shown in SEQ ID NO.1 or SEQ ID NO.

3.

2. The application according to claim 1, characterized in that, The amino acid sequence of the methyltransferase is shown in SEQ ID NO.

1.

3. The application according to claim 1 or 2, characterized in that, The Escherichia coli was ET12567.

4. The application according to claim 1 or 2, characterized in that, include: The gene encoding the methyltransferase was integrated into the genome of a methylation-deficient Escherichia coli to achieve overexpression of the methyltransferase, thereby obtaining the engineered bacteria; The amino acid sequence of the methyltransferase is shown in SEQ ID NO.1 or SEQ ID NO.

3.

5. The application according to claim 1 or 2, characterized in that, The gene encoding the methyltransferase, promoter J23119, and ribosome binding site B0034 were linked to construct an expression cassette for the methyltransferase. The expression cassette was then transformed into methylation-deficient Escherichia coli, and positive transformants were screened to obtain the engineered bacteria.

6. The application according to claim 5, characterized in that, The nucleotide sequence of the gene encoding the methyltransferase is shown in SEQ ID NO.2 or SEQ ID NO.

4.

7. A method for improving the transformation efficiency of exogenous genes in Polysporus sphaeroides, characterized in that, include: The exogenous gene is introduced into the engineered bacteria according to any one of claims 1-3 and methylated to obtain the methylated exogenous gene; The methylated exogenous gene was introduced into the *Sacchariformis*.

8. The method according to claim 7, characterized in that, The process of introducing a foreign gene into the engineered bacteria according to any one of claims 1-3 and methylating it to obtain a methylated foreign gene includes the following steps: A recombinant vector including the exogenous gene was constructed, and the recombinant vector was introduced into the engineered bacteria for methylation modification. Plasmids were extracted from the engineered bacteria to obtain methylated recombinant vectors.

9. The method according to claim 8, characterized in that, The methylated recombinant vector was introduced into the Polysporus spp. using protoplast transformation.

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