CN115873775A

CN115873775A - Method for improving secondary metabolic capacity of actinomycetes based on regulatory protein BldD posttranslational modification

Info

Publication number: CN115873775A
Application number: CN202211598999.5A
Authority: CN
Inventors: 叶邦策; 尤迪; 符瑜
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2022-12-12
Filing date: 2022-12-12
Publication date: 2023-03-31
Anticipated expiration: 2042-12-12
Also published as: CN115873775B

Abstract

The invention discloses a method for improving secondary metabolic capacity of actinomycetes based on post-translational modification of regulatory protein BldD, and belongs to the field of synthetic biology and molecular biology. According to the invention, through researching the mechanism of influence of acetylation of specific sites of transcriptional regulatory protein BldD on the functions of the transcriptional regulatory protein BldD, strain transformation is carried out by utilizing a post-translational modification metabolic engineering strategy, and the synthesis capacity of secondary metabolites of actinomycetes is improved; the regulatory protein BldD is a leading regulatory protein which is highly conserved in actinomycetes and regulates morphological differentiation and synthesis of secondary metabolites; the specific site is a lysine site in the BldD functional domain, which can be subjected to post-translational modification such as acetylation. The invention is based on the principle of post-translational modification of regulatory protein BldD, and realizes the purpose of further improving the synthesis yield of secondary metabolites on the basis of the synthesis capability of the original products.

Description

Method for improving secondary metabolic capacity of actinomycetes based on regulatory protein BldD posttranslational modification

Technical Field

The invention relates to the field of synthetic biology and molecular biology, in particular to a method for improving secondary metabolic capacity of actinomycetes based on post-translational modification of regulatory protein BldD.

Background

BldD, as an important global regulatory factor in actinomycetes, controls many genes related to growth, differentiation and secondary metabolism in actinomycetes. The BldD protein is found in streptomyces coelicolor to be a small DNA binding protein formed by encoding bldD gene, which can not only repress self expression but also repress the expression of sigma factor important in development stage, thereby influencing the morphological differentiation of streptomyces and the generation of antibiotics. It can also form hexamer with c-di-GMP, and enhance its binding ability and ability to transcriptionally regulate related genes by c-di-GMP. In addition to controlling growth and differentiation, bldD controls the expression of secondary metabolite synthase genes, such as in saccharopolyspora erythraea, which activate the expression of erythromycin synthase genes. Several studies have shown that BldD controls the synthesis of secondary metabolites such as antibiotics in various actinomycetes, and overexpression thereof can improve the synthesis level of the corresponding products, such as in streptomyces linkensis or saccharopolyspora erythraea, but there is no study on the improvement of the corresponding secondary metabolic capability by modifying BldD.

Protein acylation, an important post-translational modification, affects protein activity by changing protein structure, charge, and the like, and plays a role in transcriptional regulation, central metabolism, cellular localization, and the like. And regulating and controlling protein expression through post-translational modification. However, with the development of synthetic biology and molecular biology, the demand for synthetic engineering is increasing, and the lack of new engineering targets is a problem that afflicts many people. Therefore, the principle of acylation modification is utilized to modify regulatory proteins such as BldD and the like to search for a new modification target, and the method is very important for improving the synthesis efficiency of a target product.

Disclosure of Invention

The invention aims to provide a method for improving the secondary metabolic capacity of actinomycetes based on post-translational modification of regulatory protein BldD, which aims to solve the problems in the prior art, and realizes the purpose of improving the synthesis capacity of the secondary metabolite of actinomycetes by researching the mechanism of influence of acetylation of a specific site of transcriptional regulatory protein BldD on the function of transcriptional regulatory protein BldD and utilizing a post-translational modification metabolic engineering strategy to modify strains.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a method for improving secondary metabolic capacity of actinomycetes based on post-translational modification of regulatory protein BldD, which comprises the step of carrying out acylation modification on a specific site of the regulatory protein BldD in the actinomycetes by utilizing the post-translational modification, so as to improve the synthetic capacity of a secondary metabolite of the actinomycetes. More preferably, the regulatory protein BldD is a dominant regulatory protein highly conserved in actinomycetes that regulates morphological differentiation and synthesis of secondary metabolites. The actinomycete is a strain containing a transcription regulatory protein BldD.

Preferably, the specific site of the regulatory protein BldD is acylated by an enzymatic or non-enzymatic reaction.

Preferably, the specific site includes a lysine site in the BldD functional domain of the regulatory protein that can be acylated.

Preferably, the amino acid sequence of the regulatory protein BldD is shown in SEQ ID NO: 1:

MGDYAKALGGKLRAIRQQQGLSLHGVEQKSGGRWKAVVVGSYERGDRAVTV QKLAELADFYGVPVAELLPEGRVPSGAEPATKVVINLERLQQLPAEKVGPLARYAATI QSQRGDYNGKVLSIRTEDLRSLAIIYDMTPGELTEQLIDWGVLPPEARPAREE。

preferably, the specific site includes lysine at position 11 in the amino acid sequence shown in SEQ ID NO. 1.

Preferably, the acylation at the specific site comprises acetylation of lysine at position 11 in the amino acid sequence shown in SEQ ID NO. 1.

Preferably, the modification of the actinomycetes is realized by integrating a coding gene for mutating acetylated lysine in the regulatory protein BldD to arginine at a fixed point into a genome in the actinomycetes and removing the influence of post-translational acetylation modification on the transcriptional activity of the regulatory protein BldD, so that the synthetic capacity of a secondary metabolite of the actinomycetes is improved.

Preferably, the coding gene is as shown in SEQ ID NO:3, showing:

atgggcgactacgccaaggcgctgggcggcaGgctccgcgctatccgccagcagcaaggtctctcgctgcacggcgtcgagcagaagtcaggcgggcggtggaaggccgtggtcgtcgggtcctatgaacgtggcgaccgcgcggtgaccgtgcagaagctggccgagctggccgacttctacggggtcccggtcgcggagctgctcccggagggccgggtgccttccggcgccgagcccgccaccaaagtcgtgatcaacctggagcggctgcaacagctcccggccgagaaggtgggcccgctggcccgctacgcggccaccatccagagccagcgcggcgactacaacggcaaggtgctgtccatccgcaccgaggacctgcgatccctggccatcatctacgacatgacgcccggagagctcaccgagcagctaatcgactggggcgtgcttccgcccgaagcgcgcccggcccgggaggagtga。

SEQ ID NO:3 (bldD) ^K11R ) The amino acid sequence is shown as SEQ ID NO:2, and the following steps:

MGDYAKALGGRLRAIRQQQGLSLHGVEQKSGGRWKAVVVGSYERGDRAVTV QKLAELADFYGVPVAELLPEGRVPSGAEPATKVVINLERLQQLPAEKVGPLARYAATI QSQRGDYNGKVLSIRTEDLRSLAIIYDMTPGELTEQLIDWGVLPPEARPAREE。

the present invention uses the genetic engineering method to site-specifically mutate the acylated lysine (such as the conserved lysine site KLRAIR, etc.) of the BldD protein into arginine (simulating the lysine site which can not be acylated, and the acylation level is zero) or glutamine (simulating the lysine acylation site, and the acylation level is 100%). Acylated lysine of the BldD protein mutated to glutamine (BldD) ^K11Q ) The corresponding amino acid sequence is shown as SEQ ID NO:4, and (2) is as follows:

MGDYAKALGGQLRAIRQQQGLSLHGVEQKSGGRWKAVVVGSYERGDRAVTV QKLAELADFYGVPVAELLPEGRVPSGAEPATKVVINLERLQQLPAEKVGPLARYAATI QSQRGDYNGKVLSIRTEDLRSLAIIYDMTPGELTEQLIDWGVLPPEARPAREE。

bldD ^K11Q the nucleotide sequence of the coding gene of (1) is shown in SEQ ID NO: and 5, as follows:

atgggcgactacgccaaggcgctgggcggcCagctccgcgctatccgccagcagcaaggtctctcgctgcacggcgtcgagcagaagtcaggcgggcggtggaaggccgtggtcgtcgggtcctatgaacgtggcgaccgcgcggtgaccgtgcagaagctggccgagctggccgacttctacggggtcccggtcgcggagctgctcccggagggccgggtgccttccggcgccgagcccgccaccaaagtcgtgatcaacctggagcggctgcaacagctcccggccgagaaggtgggcccgctggcccgctacgcggccaccatccagagccagcgcggcgactacaacggcaaggtgctgtccatccgcaccgaggacctgcgatccctggccatcatctacgacatgacgcccggagagctcaccgagcagctaatcgactggggcgtgcttccgcccgaagcgcgcccggcccgggaggagtga。

the invention discloses the following technical effects:

the research of the invention finds that BldD has a plurality of acetylation sites, and the acetylation of different lysine sites can influence the structure and the function of the BldD. Acetylation in vitro and in vivo is simulated to find that lysine acetylation can weaken the capacity of BldD to form a dimer and combine with target gene DNA, and further inhibit the regulation of BldD on the transcription of downstream secondary metabolic genes, so that the production capacity of actinomycetes is limited. Based on the mechanism, the lysine site of BldD is transformed into arginine which has positive charge and cannot be acetylated, so that the acetylation limitation of the in-vivo environment on BldD is weakened, the transcription level of a product synthetic gene is improved, and the yield of a corresponding product is further improved. Therefore, the invention utilizes the principle of acylation modification to modify the BldD regulatory protein, can provide more effective modification targets on the basis of past research, and improves the synthesis efficiency of products.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows the acetylation of BldD in vivo and in vitro; (a) acetylation of BldD in vivo; (B) acetylation of BldD in vitro with treatment with AcP or AcuA;

FIG. 2 is a comparison of the mass spectral identification of acetylation sites of wild-type BldD, bldD after AcP treatment and BldD after AcuA treatment;

FIG. 3 is an in vivo mass spectrometric identification of K11 acetylation sites;

FIG. 4 shows the binding of various BldD and BldD mutants to DNA (A) BldD, bldD ^K11Q And BldD ^K11R Comparison with DNA binding; (B) BldD，BldD ^K11Q And BldD ^K11R Comparison with DNA binding with 1. Mu.M c-di-GMP addition;

FIG. 5 is a BldD homology alignment among different actinomycetes; (a) BldD homology alignment in different bacteria; (B) Sequence conservation comparison of BldD in Saccharopolyspora erythraea, streptomyces coelicolor and Streptomyces venezuelae;

FIG. 6 shows WT, obldD ^K11Q And ObldD ^K11R Comparison of the transcription levels of the different erythromycin synthetic genes in the four strains; wherein S1-S4 in the abscissa are WT, obldD ^K11Q And obddD ^K11R Four kinds of thalli;

FIG. 7 shows WT, obldD ^K11Q And obddD ^K11R Comparing the erythromycin synthesizing capacity of the four thalli; (A) comparing the yield of erythromycin from different thalli; (B) Bacillus subtilis is used as an indicator bacterium, and inhibition zones of different thalli for producing erythromycin are compared.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

1. Plasmid construction

A bldD fragment containing a homologous sequence constructed using primers pET-2077F/R (pET-2077F. A fragment his-BldD-his was amplified from plasmid pET28a-T7-his-BldD-his by using primers pIB-2077F/R (pIB-2077F.

The enzyme digestion system is as follows: mu.L Buffer, 4. Mu.L EcoRI restriction enzyme, 4. Mu.L Hind III restriction enzyme, 30. Mu.L pET28a plasmid and 7. Mu.L ddH ₂ And (O). And (3) performing gel recovery on the enzyme digestion product through agarose electrophoresis, and recovering the well cut linear plasmid by using a gel recovery kit.

The target gene is amplified by a PCR method. The PCR reaction system is as follows: 25. Mu.L of buffer, 1. Mu.L of dNTP, 1. Mu.L of forward primer, and,1 μ L of downstream primer, 1 μ L of template, 1 μ L of high fidelity DNA polymerase and 20 μ L of ddH ₂ And (O). The PCR reaction program is: pre-denaturation at 95 ℃ for 10 minutes, denaturation at 95 ℃ for 15 seconds, annealing at 56 ℃ for 15s, extension at 72 ℃ for 1 minute/1000 bp, circulating the process from denaturation to extension for 34 times, extending at 72 ℃ for 5 minutes after the circulation is finished, and finally cooling to 12 ℃. After agarose gel electrophoresis, the PCR product is compared with a marker in size, and after the PCR product is correct in size, the PCR product is purified and recovered by using a gel recovery kit.

Connecting the obtained enzyme digestion plasmid with the amplified target fragment through a multi-fragment homologous recombinase, wherein the recombination reaction conditions are as follows: 6 mu L of homologous recombinase, 4 mu L of target fragment and 2 mu L of enzyme digestion plasmid are uniformly mixed, then the mixture is bathed for 30min at the temperature of 50 ℃, after the mixture is placed on ice for 5min, all the systems are added into DH5 alpha/BL 21 competence and are gently and uniformly mixed, after the mixture is placed on ice for 30min, the mixture is thermally shocked in water bath at the temperature of 42 ℃ for 90 s, after the mixture is placed on ice for 3 min, 750 mu L of LB culture solution is added, and the mixture is activated for 45 min in a shaking table at the temperature of 37 ℃ and the rpm of 220; and coating the activated bacterial liquid on an LB solid medium plate containing 50 mu g/mL kanamycin, culturing for 12-18 hours at 37 ℃ overnight, selecting a monoclonal for sequencing, comparing with a designed sequence, and reserving the monoclonal with correct sequencing to obtain transformants containing pIB139-PermE-his-BldD-his and pIB139-PermE-his-BldD-his plasmids.

2. The over-expression plasmid is transferred into saccharopolyspora erythraea

Extracting a plasmid: 50 mu L of DH5 alpha colibacillus liquid containing over-expression plasmid is added into a 5mL LB liquid medium test tube, shaking culture is carried out for 24 hours at 37 ℃ and 220rpm, and the plasmid is extracted and purified by the cultured liquid through a plasmid extraction kit.

Plasmid transformation into saccharopolyspora erythraea: adding 50 μ L pIB139-PermE-his-BldD-his plasmid and site mutation plasmid into 50 μ L Saccharopolyspora erythraea protoplast on ice, adding 200 μ L PEG-T, mixing, and spreading all the liquid on non-resistant R ₃ Culturing at 30 deg.C for 24 hr on M solid culture medium plate, uniformly spreading 1mL apramycin liquid diluted to 125 μ g/mL on the plate, culturing at 30 deg.C for 3-5 days, spreading monoclones growing on the plate, spreading 1mL apramycin diluted to 125 μ g/mL in advance, and culturingNon-resistance R of a liquid of a mycin ₃ Culturing on an M solid medium plate at 30 ℃ for 2-3 days, picking out grown bacteria, adding the bacteria into a 5mL TSB liquid medium test tube containing 50 mu g/mL apramycin, carrying out shake culture at 30 ℃ and 220rpm for 48 hours, sucking normally grown bacteria liquid for sequencing, amplifying by M13F/R (M13F: 5- 'GTGCTGCAAGGCGATTAAGTT 3'; M13R: 5- 'TTATGCTTCCGGCTCGTATGT 3') primer, and after comparing with a designed sequence, reserving the bacteria liquid with correct sequencing to obtain a transformant containing pIB139-PermE-his-BldD-his plasmid and mutant plasmid.

3. In vitro expression and purification of proteins

And (3) culture of purified bacteria: 50 μ L of BL21 bacterial liquid with pIB139-PermE-his-BldD-his plasmid was added to a 5mL LB liquid medium tube containing 50 μ g/mL kanamycin, shake-cultured at 37 ℃ and 220rpm for 18 hours, and then the cultured bacterial liquid was added to 100mL LB liquid medium containing 50 μ g/mL kanamycin, shake-cultured at 37 ℃ and 220rpm until OD ₆₀₀ 0.4-0.6, IPTG was added to a concentration of 96. Mu.g/mL, and the mixture was transferred to a shaker at 220rpm at 20 ℃ for overnight culture.

Protein in vitro purification: after the culture is finished, the thalli are centrifuged and washed by PBS buffer solution, then the thalli are crushed by an ultrasonic crusher under the condition of low temperature, so that protein is dissolved in the PBS buffer solution, and impurities after the crushing are removed by centrifugation. Purifying the protein by using a nickel column, washing and balancing the nickel column by using 10mL of NPI-10, adding the crushed and centrifuged supernatant into the nickel column through a 0.45 mu m filter head, controlling the flow rate at 15mL/h, washing the unbound hetero protein in the nickel column by using 10mL of NPI-20, eluting the target protein by using 5mL of NPI-250, finally washing the nickel column by using 5mL of 0.5M NaOH, and preserving the nickel column by using 20mL of NPI-10. The size of the eluted target protein is verified by SDS-PAGE electrophoresis, and after the size is confirmed to be correct, the target protein is stored in PBS buffer solution by an ultrafiltration method for subsequent use.

Ultrafiltration of the protein solution: selecting an ultrafiltration tube with the aperture about 1/3 of the size of the target protein for ultrafiltration, and replacing the protein buffer solution with PBS; firstly, filling deionized water in an ultrafiltration tube, centrifuging for 10-15 minutes at 4 ℃ and 3000g, cleaning the ultrafiltration tube, simultaneously observing whether the ultrafiltration tube is intact, and then pouring off the deionized water; filling the ultrafiltration tube with PBS buffer solution, centrifuging at 4 deg.C for 10-15 min at 3000g, and further cleaning the ultrafiltration tube; adding protein solution, filling with PBS buffer solution, centrifuging at 4 deg.C to obtain residual liquid with volume of about 1mL, filling with PBS buffer solution, centrifuging at 4 deg.C to obtain residual liquid with volume of 500-1000 μ L, and taking out the protein solution for subsequent experiment.

SDS-PAGE electrophoresis is adopted, and a gel imager is used for photographing after decoloration.

4. Western-blot experiment

The purified BldD protein was quantitated and used in the AcP acetylation assay in the following reaction system:

TABLE 1 reaction System

The method comprises the steps of carrying out an experiment by using protein gel after electrophoresis, clamping the protein gel in a sandwich splint according to the sequence of positive pole-sponge-filter paper-PVDF membrane-protein gel-filter paper-sponge-negative pole, putting the assembled splint into an electrophoresis tank filled with a membrane transfer buffer solution, adding an ice bag to ensure that the experiment is carried out under a low temperature condition, carrying out electrophoresis for a certain time under the voltage of 100V (the size of target protein determines the electrophoresis time, 1min/KD is determined in principle, but the electrophoresis time is preferably not less than 20min and not more than 90 min), stopping electrophoresis, taking out the PVDF membrane, enabling the surface contacting with the protein gel to face upwards, washing the PVDF membrane with 10mL TBST buffer solution for three times, adding 10mL of confining liquid (5% ABV is dissolved in TBST) after the completion of washing, carrying out sealing for 2 hours, adding a certain amount of primary antibody, carrying out overnight incubation at 4 ℃, pouring the confining liquid, washing with 10mL of TBST for 3 times, adding 10mL of TBST and a certain amount of secondary antibody after the completion of the washing, carrying out incubation for 1 hour at normal temperature, carrying out color development in an ECL observation.

5. Saccharopolyspora erythraea culture method

Inoculating 50 μ L of preserved Saccharopolyspora erythraea bacterial solution into 5mL TSB liquid test tube, adding glass beads into the test tube, shake culturing at 30 deg.C and 220rpm for 48 hr, collecting 500 μ LInoculating the bacterial liquid cultured in the L test tube into 30mL of TSB liquid culture medium, adding glass beads into a shake flask in advance, carrying out shake-bed culture at 30 ℃ and 220rpm for 48 hours, and finally inoculating 0.5-1 mL of the seed liquid into a 500mL triangular flask containing 50mL of TSB culture medium to ensure that the initial OD is increased ₆₀₀ 0.05, and was subjected to shake culture at 30 ℃ and 220 rpm.

6. Acetylation site identification

In vitro acetylation site identification: mu.L of purified BldD protein was mixed with 10. Mu.L of acetyl phosphate (AcP) (100 mM), control 10. Mu.L of ultrapure water was incubated at 37 ℃ for 5 hours, and proteins were analyzed for acetylation in vitro by SDS-PAGE and Western-blot, and after confirmation of acetylation, proteins were separated by SDS-PAGE electrophoresis and cleaved from the gel for mass spectrometric identification.

In vivo acetylation site identification: the cultured saccharopolyspora erythraea is transferred into a centrifuge tube, centrifuged and washed once with PBS buffer, and then resuspended in PBS buffer and added with protease inhibitor and histone deacetylase inhibitor (100. Mu.M trichostatin A,50mM nicotinamide and 50mM sodium butyrate). The bacterial cells are crushed by an ultrasonic crusher and centrifuged to remove cell debris, and the supernatant is the holoprotein solution. A cellular holoprotein solution containing about 300 mu G of protein is taken, 5 mu G of BldD antibody is added, the cell is incubated for 2 hours at 4 ℃, then 40 mu L of protein A + G agarose gel is added, the cell is incubated overnight at 4 ℃, after the incubation is finished, the gel is washed for 5 times by PBS buffer solution, finally, the protein combined with the gel is eluted in SDS loading buffer solution by boiling, and the protein acetylation level is analyzed by SDS-PAGE protein electrophoresis and Western-blot. The correctly sized bands were excised from the SDS-PAGE gel and the acetylation sites were mass-analysed.

7. Protein spot mutation

Point mutation is carried out on bldD in pET28a-T7-his-BldD-his plasmid by using a point mutation kit and primers K11Q F/R and K11R F/R, a gene sequence aag corresponding to 11-bit lysine is mutated into a cag sequence and an agg sequence, namely, lysine is mutated into glutamine and arginine respectively, then the mutated plasmid is transformed into BL21 competence, and the transformation and subsequent steps are the same as those before the mutation.

K11Q F：5’-GCGGCCAGCTCCGCGC-3’；

K11Q R：5’-GGCCGCCCAGCGCCTT-3’；

K11R F：5’-GCGGCAGGCTCCGCGC-3’；

K11R R：5’-CTGCCGCCCAGCGCCT-3’。

8. Blocking gel electrophoresis analysis (EMSA)

Preparation of probes for blocking gel electrophoresis experiments: the preparation of the probe was completed by amplifying the desired probe using a synthetic biotin-labeled primer (Shanghai Bioengineering Co., ltd.). The amplification primers are as follows:

bldD_F：5’-AGCCAGTGGCGATAAGTCCCGACGGCTGTCGGC-3’；

bldD_R：5’-AGCCAGTGGCGATAAGCCGTTTCATTCGCCCCGTC-3’。

retardation gel test: diluting protein to required gradient concentration, adding 4 mu L of protein solution (no protein tube is replaced by deionized water) into an EP tube, covering a cover with 2 mu L of deionized water and 2 mu L of 5 × EMSA combined buffer solution, incubating at normal temperature for 15 minutes, adding 1 mu L of diluted probe solution, mixing uniformly, and incubating at low temperature of 18 ℃ for 30 minutes; preparing 20mL of non-denatured glue for EMSA experiments (1.0mL of 10 xTBE buffer,14.2mL of ddH2O,4mL of 39; adding all incubated samples into gel holes after pre-electrophoresis, putting an ice bag into an electrophoresis tank, performing electrophoresis for 1.5-2 hours at low temperature by using 100V voltage, stopping electrophoresis after strips reach proper positions, taking out non-denatured gel for performing membrane conversion experiment by using N ⁺ The steps of the nylon membrane are the same as those of Western-blot, the membrane transferring buffer solution is 0.5 xTBE buffer solution, an ice bag is placed in an electrophoresis tank, the membrane is transferred for 30-60 min under the condition of 380mA constant current at low temperature, after the membrane transferring is finished, the surface of the nylon membrane contacting with the EMSA adhesive faces upwards, and the nylon membrane is placed in a clean containerIn the method, an ultraviolet cross-linking instrument (UV-light cross-linker) is used, the ultraviolet wavelength of 254nm is selected, the nylon membrane is cross-linked for 1 minute, the cross-linked nylon membrane is sealed by 15mL of sealing solution at normal temperature for 30 minutes, after sealing is completed, 15mL of sealing solution and 5 mu L of Streptavidin-HRP Conjugate (1.

To analyze the effect of BldD acetylation on actinomycete metabolism in vivo, bldD was included, and BldD was included ^K11Q ，BldD ^K11R Recombinant plasmids of the gene fragments were introduced into Saccharopolyspora erythraea to overexpress these genes in the cells. The cultured cells were collected and subjected to RT-PCR. The method comprises the following specific steps:

and (3) taking the thalli at the corresponding time point, extracting RNA in the thalli by using an RNA extraction kit, and then reversely transcribing the extracted RNA into cDNA by using an RNA reverse transcription kit. The cDNA was diluted to 100 ng/. Mu.L by nucleic acid-free water, the sample was applied in a 20. Mu.L system, and RT-PCR experiments were performed using the RT-PCR primers corresponding to the primers, according to the procedure set forth in the specification.

PCR System (20. Mu.L): SYBR Premix Ex Taq GC (2X) 10. Mu.L; PCR Forward Primer (10. Mu.M) 0.5. Mu.L; PCR Reverse Primer (10. Mu.M) 0.5. Mu.L; template cDNA (. About.100 ng) 1. Mu.L; 8 μ L of sterile distilled water.

PCR procedure: 60s at 95 ℃;95 ℃ for 10s,60 ℃ for 10s,72 ℃ for 30s,40cycles; 5s at 65 ℃.

The gene primers designed by the RT-PCR detection are as follows:

SACE_8101-F：5’-GTTGCGATGCCGTGAGGT-3’；

SACE_8101-R：5’-CGGGTGTTACCGACTTTCA-3’；

eryBIV-F：5’-GCAGCCGCAGGATCACGC-3’；

eryBIV-R：5’-GCCGCCCGTGTTGCTCTA-3’；

eryAI-F：5’-CCGCTGATGCCGAACGAC-3’；

eryAI-R：5’-CACCCTTCCCCGCACTCTG-3’；

ermE-F：5’-CCTCCAGGCACCAGTCCAC-3’；

ermE-R：5’-AGTCGTTGCGGGAGAAGCT-3’；

eryK-F：5’-CCGATGGACCACGAGCAGTT-3’；

eryK-R：5’-AAGGCGGGAGATCAGGTCGT-3’。

to investigate whether the acetylation mechanism of BldD could be exploited to engineer the protein to increase the concentration of related secondary metabolites, four strains (WT, obldD) were further planned to be tested after finding that mutation of KLRAIR to arginine at the lysine site could further increase the expression of the erythromycin synthesis gene ^K11Q ，ObldD ^K11R ) The product concentration of (2). Respectively carrying out fermentation culture on the four strains in a TSB culture medium for 120h, extracting supernatant, and respectively measuring the erythromycin yields of the four strains by using a zone of inhibition experiment and an HPLC experiment which take bacillus subtilis as an indication, wherein the method specifically comprises the following steps:

(1) Zone of inhibition experiment

50 μ L of Bacillus subtilis was inoculated into 5mL of LB liquid medium and cultured at 37 ℃ and 220rpm to OD ₆₀₀ About 0.6. Mu.L of the bacterial solution was uniformly applied to the surface of LB solid medium plate. Wild type, obldD were cultured using 50mL of TSB broth ^K11Q And ObldD ^K11R Centrifuging at 4 deg.C and 5600rpm to collect supernatant, dripping 10 μ L of supernatant into different regions of the same LB plate coated with Bacillus subtilis, drying, culturing in 37 deg.C incubator for 6-8 hr, and measuring the inhibition zones with vernier caliper to compare the inhibition activities of erythromycin.

(2) HPLC determination of erythromycin concentration

After fermentation, the fermentation liquor is subjected to constant volume to the same volume, is centrifuged for 10min at 8000 Xg, and supernatant with the same volume (50 mL) is collected and frozen at-80 ℃, is freeze-dried to powder by a freeze dryer, is added with a certain amount of acetonitrile to be fully dissolved, is centrifuged to collect supernatant, and is dried by a speedVac of a centrifugal concentrator. Then dissolved in 1mL of methanol and filtered through a 0.22 μm organic phase filter before injection. Erythromycin standards (gradient dilutions) were prepared at concentrations of 2.5mg/mL, 1.25mg/mL, 0.625mg/mL, 0.3125mg/mL, 0.15625mg/mL, and 0.078125mg/mL, respectively, and used to prepare calibration curves after measurement. Erythromycin samples and standards were analyzed by HPLC.

The instrument model is as follows: agilent 1100HPLC;

a chromatographic column: c18 column (5 μm inner diameter,250 × 4.6 mm);

wavelength: 215nm;

sample introduction amount: 10 mu L of the solution;

mobile phase: 45% phase A: 30mM KH ₂ PO ₄ (pH 8.0); 55% of phase B: acetonitrile;

flow rate: 1mL/min.

9. Results and analysis

(1) Acetylation of KLRAIR at the lysine site affected the binding of BldD to the DNA site

It was found that in vitro BldD could be acetylated by AcP and also by the saccharopolyspora erythraea acetyltransferase AcuA dependent enzymatic reaction by incubating 10mM AcP with BldD at 37 deg.C (FIG. 1B). However, bldD was also acetylated in vivo as detected by enrichment in BldD in vivo and acetylation (fig. 1A). It was found by mass spectrometric identification that in vitro multiple sites of BldD could be acetylated, whereas under in vivo conditions only the lysine site KLRAIR was acetylated (fig. 2 and fig. 3). These results indicate that BldD has a phenomenon of acetylation at the lysine site KLRAIR depending on AcP catalysis in the thallus.

In order to investigate whether acetylation of the lysine position KLRAIR has an effect on the function of BldD, the lysine position KLRAIR was mutated to glutamine (Q) which has no positive charge and to arginine (R) which has a positive charge but cannot be acetylated, as exemplified by the lysine position KLRAIR. EMSA experiments were performed with these two mutant proteins and wild-type BldD with DNA probes. The results of the experiments show that the binding capacity of BldD to DNA and c-di-GMP is impaired after mutation of lysine to Q, while the DNA binding capacity of BldD mutated to R is not much affected (FIGS. 4A and B). These experimental results indicate that acetylation of the lysine site KLRAIR inhibits the binding of BldD to DNA and c-di-GMP. The binding to DNA or c-di-GMP is critical in the functioning of BldD, and when it is affected, the physiological function of BldD must also be affected.

(2) The modification aiming at the BldD lysine site of Saccharopolyspora erythraea is also suitable for other actinomycetes

As Saccharopolyspora erythraea BldD is selected as a research object in the research process, in order to prove that the corresponding research and application can be applicable to other actinomycetes, the universality and the conservation of BldD in different actinomycetes are analyzed. Analysis of the evolutionary tree shows that BldD is widely distributed in various actinomycetes and has high homology, and compared with the sequences of BldD of Streptomyces coelicolor, streptomyces venezuelae and Saccharopolyspora erythraea, the sequences of BldD are highly conserved (FIG. 5). Most of the lysine that can be acetylated in BldD are located in a conserved sequence, wherein K11 of Saccharopolyspora erythraea BldD, namely a conserved lysine site KLRAIR, is also present in other actinomycetes and has high conservation, so that the acetylation of the site in the Saccharopolyspora erythraea BldD can be studied and utilized by other actinomycetes.

(3) The expression of the secondary metabolite synthesis gene can be further improved by modifying the corresponding sites by BldD acetylation

The results of RT-PCR experiments show that BldD is expressed compared with the over-expression of wild-type BldD ^K11Q The overexpression of (A) is difficult to effectively increase the transcriptional level of erythromycin synthesis-associated genes, while BldD ^K11R The overexpression of (2) further improves the transcription level of erythromycin synthetic gene on the basis of wild-type BldD overexpression thalli (FIG. 6). The experimental results show that: in the thallus, acetylation of lysine site KLRAIR can inhibit the control capacity of BldD on downstream genes, and the BldD is over-expressed ^K11R The inhibition effect can be effectively shielded, and the transcription level of the secondary metabolite synthetic gene can be further improved on the basis of wild over-expression strains.

(4) The improvement aiming at the BldD acetylation site can further improve the synthesis capacity of the secondary metabolite of the thallus

Results of the bacteriostatic circle experiment and the HPLC experiment show that: of these four strains, obldD ^K11R The erythromycin has the largest inhibition zone and the highest potency, the titer is the highest, the titer is the second order of ObldD, and then the titer is the second order of ObldD ^K11Q And WT (fig. 7). Shows that the capacity of producing erythromycin by saccharopolyspora erythraea can be further improved and the yield of products can be improved by mutating KLRAIR at a lysine site of bldD into arginine (R). This also shows that the mutation of KLRAIR at lysine site to arginine can further improve the synthesis ability of the secondary metabolite of the bacterial cells.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A method for improving the secondary metabolic capability of actinomycetes based on post-translational modification of a regulatory protein BldD is characterized in that the method comprises the step of carrying out acylation modification on a specific site of the regulatory protein BldD in the actinomycetes by utilizing the post-translational modification, so that the synthetic capability of the secondary metabolite of the actinomycetes is improved.

2. The method of claim 1, wherein the specific site of the regulatory protein BldD is acylated by an enzymatic or non-enzymatic reaction or by point mutation.

3. The method of claim 1, wherein the specific site comprises a lysine site in the BldD functional domain of the regulatory protein that is capable of being acylated.

4. The method of claim 3, wherein the amino acid sequence of the regulatory protein BldD is set forth in SEQ ID NO 1.

5. The method according to claim 4, wherein the specific site comprises lysine at position 11 in the amino acid sequence shown in SEQ ID NO. 1.

6. The method according to claim 5, wherein the acylation at the specific site comprises acetylation of lysine at position 11 in the amino acid sequence shown in SEQ ID NO. 1.

7. The method of claim 6, wherein the modification of the actinomycete is achieved by site-directed mutagenesis of the acetylated lysine in the regulatory protein BldD into the coding gene for arginine integrated into the genome of the actinomycete to remove the effect of post-translational acetylation modification on the transcriptional activity of the regulatory protein BldD, thereby improving the synthetic capacity of the secondary metabolite of the actinomycete.

8. The method of claim 7, wherein the encoding gene is as set forth in SEQ ID NO:3, respectively.