Dynamic regulation and control system and method for actinomycete gene expression and application

Abstract

The invention discloses a dynamic regulation and control system and method for actinomycete gene expression and application. According to the invention, a dynamic regulation element for actinomycete gene expression is designed, a gene sequential expression dynamic regulation system suitable for actinomycetes is constructed based on the dynamic regulation element, and a plurality of target spots on a DNA sequence which is designed in advance and arranged in series are subjected to sequential single-base editing through a single-base editing enzyme ABE8e to finally target a promoter 35box sequence of the actinomycete gene. Finally, the expression regulation of the target gene in the actinomycetes is realized by activating or suppressing the promoter. According to the present invention, the number / length of the series Fuze-like sequence can be preset so as to control the expression time of the gene in the way of actinomycete antibiotic synthesis and the like;

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and specifically relates to a vector or expression system suitable for a prokaryotic cell host other than Escherichia coli. Background Art

[0002] As an important microbial resource, actinomycetes have important application value in the fields of medicine and biotechnology. Studies have shown that actinomycete metabolites (such as alkaloids, terpenes, esters, peptides, etc.) have significant biological activity. Modern pharmacology shows that actinomycete metabolites have anticancer, anti-tumor, antibacterial and other effects. Actinomycetes also play an important role in the research and development and production of drugs, enzyme preparations, immunomodulators, herbicides and pesticides. In addition, actinomycetes are an important source of natural antibiotics. About 4,000 natural antibiotics are produced from the genus Streptomyces, such as common anthracyclines, chloramphenicol, β-lactams, macrolides and tetracyclines.

[0003] Streptomyces is a typical actinomycete and an important industrial antibiotic producer. Among them, Streptomyces coelicolor is a model strain for studying the secondary metabolism of Streptomyces, which can produce a variety of antibiotics, such as actinorhodin (ACT), undecylprodigiosin (RED), calcium-dependent antibiotic (CDA), etc. Among them, actinorhodin is a blue pigment antibiotic. It is the first antibiotic to clone the entire biosynthetic gene cluster and one of the best model compounds for studying type II polyketide synthase (PKS) and its auxiliary enzymes and PKS post-cutting enzymes. However, the antibiotic synthesis level of wild Streptomyces isolated from nature is very low, which is difficult to meet the requirements of industrialization; the already industrialized engineered strains also need to continuously improve production to reduce production costs.

[0004] In order to rationally regulate metabolic pathways to improve the production capacity of microorganisms, static regulation methods such as overexpression of key genes, inhibition or knockout of bypass competitive metabolic pathways are generally used to strengthen the target product synthesis metabolic flow to improve the production performance of strains. However, static regulation methods ignore the dynamic adjustability of the overall metabolic network and are difficult to adapt to complex and changeable culture conditions. In addition, the synthesis of target products is often closely related to fatty acid synthesis, reducing power balance, energy metabolism and cell growth. It is difficult to maximize industrial output through simple static regulation alone. Therefore, the dynamic adjustability of the metabolic network is an important factor in system stability. Only a dynamic regulation system that can respond to environmental changes in real time can achieve stable growth, reproduction and production of microorganisms under complex conditions. How to establish a dynamic regulation system to maximize the synthesis of target products based on dynamic balance between factors such as cell growth is a hot and difficult issue in current research.

[0005] Like most microorganisms, Streptomyces undergoes a transition from primary metabolism (logarithmic growth phase) to secondary metabolism (plateau phase) during the late fermentation process. The premature expression of secondary metabolites, especially antibiotics, will seriously affect the natural growth of Streptomyces in the early fermentation period. The mismatch between the synthesis sequence of secondary metabolites and the physiological state of Streptomyces precursor supply is an important factor restricting the increase in the yield of secondary metabolites such as antibiotics. Studying the regulatory technology system and methods for gene expression in metabolic pathways of industrial actinomycetes such as Streptomyces is the key to improving the ability of actinomycete drug synthesis systems. At present, artificial regulation of actinomycetes at the gene transcription level mainly adopts static regulation strategies such as regular addition of inducers or repressors, promoter engineering or CRISPRa / i, which easily lead to imbalance and disorder of bacterial metabolism. Therefore, it is necessary to develop a dynamic regulatory system to transform the metabolism of industrial actinomycetes and optimize the fermentation process to increase the yield of the target product. However, the Streptomyces genome is rich in secondary metabolic gene clusters and regulatory genes. Genes related to a specific secondary metabolic pathway in Streptomyces are usually present in the form of gene clusters on linear chromosomes. The Streptomyces genome has a high GC content and complex transcriptional regulation. The promoters of Streptomyces also show great heterogeneity, that is, promoters that are generally available in other microorganisms often do not show activity in Streptomyces. There are few free plasmid vectors available for actinomycetes such as Streptomyces. The genetic operating system of actinomycetes such as Streptomyces is mainly achieved through integrated plasmids. At present, there are no relevant literature and patent reports on the dynamic regulation system of gene expression for actinomycetes. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a system and method for dynamic regulation of actinomycete gene expression and its application, so as to achieve dynamic regulation of temporal gene expression of actinomycetes.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A dynamic regulatory switch element for actinomycete gene expression, comprising a regulatory element for turning on downstream gene expression or a regulatory element for turning off downstream gene expression, wherein:

[0009] The overall sequence composition of the regulatory element for turning on downstream gene expression is as follows:

[0010] 5'-TacDD / TacDC promoter sequence-stimulation target sequence-tandem DNA sequence basic unit × n-NBB-TGG-3';

[0011] The overall sequence composition of the regulatory element for shutting down downstream gene expression is as follows:

[0012] 5'-KasOpCD promoter sequence-suppression target sequence-tandem DNA sequence basic unit × n-NBB-TGG-3';

[0013] Wherein, N represents any base, B represents one of the bases G, T, and C, and n represents the number of basic units, n = 1, 2, 3, ...;

[0014] Among them, the TacDD promoter sequence is shown as SEQ ID NO.1, the TacDC promoter sequence is shown as SEQ ID NO.2; the KasOpCD promoter sequence is shown as SEQ ID NO.3; the tandem DNA sequence basic unit is shown as SEQ ID NO.4; the excitation target sequence is shown as SEQ ID NO.5, and the suppression target sequence is shown as SEQ ID NO.6.

[0015] In some specific examples of the present invention, {tandem DNA sequence basic unit × n-NBB-TGG} includes 1 initial transduction sequence and n-1 transduction sequences in the initial state, the initial transduction sequence is composed of the tandem DNA sequence basic unit-NBB-TGG, the transduction sequence is composed of the tandem DNA sequence basic unit-NBB-TAA, the 5' end of the target of the initial transduction sequence and the 3' end of the target of the transduction sequence have an overlapping region (5'-NBB-TAA-3'), the adjacent 5' ends and 3' ends of the targets of any two transduction sequences have an overlapping region (5'-NBB-TAA-3'), the target of the initial transduction sequence and the target of the transduction sequence share a transduction sgRNA, and the nucleotide sequence of the transduction sgRNA is shown in SEQ ID NO.8.

[0016] In some specific examples of the present invention, the sgRNA used for the excitation target / suppression target is called excitation / suppression sgRNA, the nucleotide sequence of the excitation sgRNA is shown in SEQ ID NO.9, and the nucleotide sequence of the suppression sgRNA is shown in SEQ ID NO.10.

[0017] At the same time, the present invention also provides a dynamic regulation system for actinomycete gene expression, including: a single base editing enzyme expression plasmid pCRISPR-PtipA-streABE8e plasmid and a pSOK804E-Fuze plasmid containing a Fuze-like sequence;

[0018] The pCRISPR-streABE8e plasmid is obtained by the following method: replacing ABE7.10 of pCRISPR-aBEST with codon-optimized TadA8e to obtain pCRISPR-PtipA-ABE8e-nCas9; and then performing a point mutation D10A on nCas9 of pCRISPR-PtipA-ABE8e-nCas9 to obtain the pCRISPR-PtipA-streABE8e plasmid;

[0019] The pSOK804E-Fuze plasmid is obtained by the following method: cloning the dynamic regulatory switch element, actinomycete target gene, conduction sgRNA and stimulation / suppression sgRNA as described in any one of claims 1 to 3 into the pSOK804E plasmid to obtain the pSOK804E-Fuze plasmid.

[0020] In some specific examples of the present invention, the target gene includes a functional gene or a regulatory factor.

[0021] In some specific examples of the present invention, the target gene includes a functional gene or a regulatory factor in a natural product synthesis metabolic pathway or an antibiotic synthesis metabolic pathway.

[0022] In some specific examples of the present invention, the target gene is sco5087.

[0023] In addition, the present invention also provides a method for dynamically regulating actinomycete gene expression, comprising:

[0024] (1) Designing a dynamic regulatory switch element for actinomycete gene expression according to any one of claims 1 to 3, cloning the dynamic regulatory switch element, the actinomycete target gene, the conducting sgRNA and the stimulating / repressing sgRNA into a pSOK804E plasmid, thereby obtaining a pSOK804E-Fuze plasmid;

[0025] (2) Replacing ABE7.10 of pCRISPR-aBEST with codon-optimized TadA8e to obtain pCRISPR-PtipA-ABE8e-nCas9; then performing a point mutation D10A on nCas9 of pCRISPR-PtipA-ABE8e-nCas9 to obtain pCRISPR-PtipA-streABE8e plasmid;

[0026] (3) The pSOK804E-Fuze plasmid and the pCRISPR-PtipA-streABE8e plasmid are sequentially transferred into the target actinomycetes by conjugation transfer or preparation of actinomycete protoplasts.

[0027] In some specific examples of the present invention, in step (3), the method for sequentially transferring the pSOK804E-Fuze plasmid and the pCRISPR-PtipA-streABE8e plasmid into the target actinomycetes by conjugation transfer is as follows: the Escherichia coli ET12567 bacterial solution containing the pSOK804E-Fuze plasmid is conjugated with the pre-germinated target actinomycete spores to form integrated strain spores; the Escherichia coli ET12567 bacterial solution containing the PCRISPR-PtipA-streABE8e plasmid is conjugated with the integrated strain spores to transfer the dynamic regulation system into the target actinomycetes.

[0028] In some specific examples of the present invention, the target gene includes a functional gene or a regulatory factor.

[0029] In some specific examples of the present invention, the target gene includes a functional gene or a regulatory factor in a natural product synthesis metabolic pathway or an antibiotic synthesis metabolic pathway.

[0030] In some specific examples of the present invention, the target gene is sco5087.

[0031] The present invention also discloses the application of the above-mentioned dynamic regulation switch element or the dynamic regulation system of actinomycete gene expression, including but not limited to: being used for pre-programming the temporal expression of genes synthesizing different metabolite pathways of actinomycetes; or being used for controlling the gene expression of actinomycete strains in the natural product and antibiotic synthesis industries.

[0032] The present invention completes the dynamic regulation switch element of actinomycete gene expression by designing and modifying the actinomycete promoter, codon optimization of ABE8e, and construction of a gene expression vector regulated by sequential editing of the basic unit of the tandem DNA sequence, and constructs a dynamic regulation system of actinomycete gene expression (Actinomycete Biofuze, referred to as ABioFuze). The single-base editor streABE8e performs sequential single-base editing on several target points on the pre-designed tandemly arranged DNA sequence, and finally targets and modifies the promoter-35box sequence of the actinomycete gene, so as to close or open the promoter overlapped by the last target point, and achieve the purpose of delayed regulation of the expression of the target gene. ABiofuze of the present invention can control the expression time of the target gene of actinomycetes by presetting the number (length) of tandem conduction sequences.

[0033] Compared with the prior art, the present invention has the following beneficial technical effects:

[0034] The present invention constructs a dynamic regulation system for the temporal expression of genes in actinomycetes (Actinomycete Biofuze, referred to as ABioFuze), which uses the single-base editing enzyme ABE8e to perform sequential single-base editing on several target points on a pre-designed tandemly arranged DNA sequence, ultimately targeting the promoter-35box sequence of the actinomycete gene, and finally achieving regulation of the expression of the target gene in actinomycetes by activating or inhibiting the promoter.

[0035] The ABiofuze system of the present invention can control the expression time of one or more target genes in actinomycetes by presetting the number (length) of serially connected transduction sequences, thereby achieving dynamic regulation of the temporal expression of the target genes.

[0036] The regulatory element capable of presetting the timing of gene expression realized by the present invention can preset the gene expression time in actinomycetes, and its characteristics enable it to regulate the temporal sequential expression of multiple genes through ABioFuze elements of different lengths in parallel, and can be used for the intelligent switching strategy between the "growth" mode and the "production" mode of the bacteria, and has important application value in the fields of industrial actinomycete metabolism and drug synthesis regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Comparison of promoter strength of three promoters before and after editing by ABE8e.

[0038] Figure 2 Schematic diagram of the dynamic regulatory switch elements of actinomycete gene expression.

[0039] Figure 3A This is the plasmid map of pCRISPR-CY41-cBEST.

[0040] Figure 3B Shown Figure 3A Sequence information of the sgRNA insertion region targeting the sco5087 gene in the construction of the pCRISPR-CY41-cBEST plasmid.

[0041] Figure 4 This is the plasmid map of pSOK804E-tacDC-8FuzeON-sco5087.

[0042] Figure 5 This is the plasmid map of pCRISPR-PtipA-streABE8e.

[0043] Fig. 6A This is the growth condition of mycelium without impurities after dilution and smearing on non-selective culture medium and inverted culture at 37℃ for 3 to 5 days.

[0044] Figure 6B To verify the growth of the obtained strain on MS plates containing abramycin (left) and without abramycin (right).

[0045] Figure 6C The results of mycelial PCR sequencing of the selected plasmid-removed clones are shown in Figure 2.

[0046] Figure 7 Verification principle and results of VWB integration site insertion in Streptomyces coelicolor.

[0047] Figure 8 The DNA sequencing results of the ABioFuze system (tacDC-8Fuze-sco5087) containing 8 transduction sequences were transferred into the Streptomyces Δ5087 strain on different days.

[0048] Fig. 9 The transcription level analysis of sco5087, a key gene for actinomycin synthesis, under different culture times is shown (**** in the figure indicates significant differences, P<0.0001). DETAILED DESCRIPTION

[0049] The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings and specific examples. It should be understood that these examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0050] Streptomyces coelicolor is a model strain for studying the secondary metabolism of Streptomyces. Hereinafter, we will use Streptomyces coelicolor as an example to design dynamic regulatory switch elements for actinomycete gene expression.

[0051] A dynamic regulatory switch element for actinomycete gene expression, comprising a regulatory element for turning on downstream gene expression or a regulatory element for turning off downstream gene expression, wherein:

[0052] The overall sequence composition of the regulatory element for turning on downstream gene expression is as follows:

[0053] 5'-TacDD / TacDC promoter sequence-stimulation target sequence-tandem DNA sequence basic unit × n-NBB-TGG-3';

[0054] The overall sequence composition of the regulatory element for shutting down downstream gene expression is as follows:

[0055] 5'-KasOpCD promoter sequence-suppression target sequence-tandem DNA sequence basic unit × n-NBB-TGG-3';

[0056] Wherein, N represents any base, B represents one of the bases G, T, and C, and n represents the number of basic units, n = 1, 2, 3, ...;

[0057] Among them, the TacDD promoter sequence is shown as SEQ ID NO.1, the TacDC promoter sequence is shown as SEQ ID NO.2; the KasOpCD promoter sequence is shown as SEQ ID NO.3; the tandem DNA sequence basic unit is shown as SEQ ID NO.4; the excitation target sequence is shown as SEQ ID NO.5, and the overlapping region with the promoter-35box is 5'-TATCAA-3'; the suppression target sequence is shown as SEQ ID NO.6, and the overlapping region with the promoter-35box is 5'-TGTCAA-3'.

[0058] Figure 1 The promoter strengths of the above three promoters before and after ABE8e editing are given. We constructed a series of promoter strength test vectors using pSOK804E plasmid as the backbone and eGFP as the reporter gene. Using M1142 as the reporter strain and the housekeeping gene hrdB as the internal reference, the transcription level of the eGFP gene before and after the promoter editing was tested. As can be seen from Figure 1, the promoter strength of the target sites on the tacDC and tacDD promoters was significantly improved after ABE8e editing (****, P<0.0001), and the promoter strength of the target sites on the kasOpCD promoter was significantly reduced after ABE8e editing (****, P<0.0001).

[0059] like Figure 2As shown, in the initial state, {tandem DNA sequence basic unit × n-NBB-TGG} includes 1 initial conduction sequence and n-1 conduction sequences, wherein the initial conduction sequence is located at the 3' end of the overall sequence, and its composition is a tandem DNA sequence basic unit -ACG-TGG (5'-AGCTAACTGCAGTCACT-AGC-TGG-3'); the conduction sequence is located in the middle of the overall sequence, and its composition is a tandem DNA sequence basic unit -ACG-TAA (5'-AGCTAACTGCAGTCACT-AGC-TAA-3'), the 5' end of the target of the initial conduction sequence and the 3' end of the target of the conduction sequence have an overlapping region (5'-AGC-TAA-3'), the adjacent 5' ends and 3' ends of the targets of any two conduction sequences have an overlapping region (5'-AGC-TAA-3'), the target of the initial conduction sequence and the target of the conduction sequence share a conduction sgRNA, and the sequence of the conduction sgRNA is shown in SEQ ID NO.8. Although only the target of the initial conduction sequence can be recognized and edited by ABE8e in the initial state, after the target of the initial conduction sequence is modified by ABE8e, since the 5' end of the target of the initial conduction sequence overlaps with the 3' end of the target of the conduction sequence, the above modification causes the PAM of the conduction target to be activated, and the conduction target is converted into the initial conduction target and then modified, and activates the next conduction target, and so on. The various targets of the entire sequence from the 3' end to the 5' end will be activated in sequence, and finally the -35box base of the promoter will be modified to activate or inactivate the promoter, thereby achieving the purpose of regulating gene expression.

[0060] Among them, the sgRNA used for the excitation target / suppression target is called excitation / suppression sgRNA, the nucleotide sequence of the excitation sgRNA is shown in SEQ ID NO.9, and the nucleotide sequence of the suppression sgRNA is shown in SEQ ID NO.10.

[0061] Based on the above-designed dynamic control switch element, a dynamic control system for gene expression of Streptomyces was constructed. The following will be described in detail using sco5087, a key gene for the synthesis of actinomycetoxypurpurin in Streptomyces coelicolor, as a target gene and an example of turning on the expression of the target gene. It should be pointed out that the target gene is not limited to sco5087 exemplified below, but includes functional genes or regulatory factors, especially, functional genes or regulatory factors including natural product synthesis metabolic pathways and antibiotic synthesis metabolic pathways.

[0062] 1. Raw materials and method description

[0063] E. coli DH5α competent cells and E. coli ET12567 / pUZ8002 competent cells are both commercially available products. In the following examples, E. coli DH5α competent cells were purchased from TOLOBIO (CC96102), and E. coli ET12567 / pUZ8002 competent cells were made in-house in the laboratory using the Ultra-Competent CellPreps Kit from Shanghai Shenggong Biotechnology Co., Ltd.

[0064] Streptomyces coelicolor M145 and Streptomyces coelicolor M1142 are both commercially available products. In the following examples, Streptomyces coelicolor M145 and M1142 were provided by Professor Guan Wenjun of Zhejiang University.

[0065] The DNA polymerase used for amplification was Gloria Nova HS2× Master Mix (RK20717) produced by ABclonal, and the DNA polymerase used for verification was 2× PCR SuperMix (+dye), the NcoI restriction endonuclease used to prepare the linearized vector is Takara's QuickCut TM The Nco I kit and the alkaline phosphatase used for dephosphorylation to prevent self-ligation of the linearized vector were FastAP from Thermo Fisher Scientific.

[0066] The plasmid extraction kit was purchased from AXYGEN Co., Ltd., the PCR product nucleic acid purification kit was purchased from Promega, and the cloning kit was 2×MultiF Seamless Assembly Mix (RK21020) from ABclonal.

[0067] pCRISPR-aBEST and pCRISPR-cBEST plasmids were purchased from Addgene, and pSOK804E plasmid was provided by Professor Guan Wenjun of Zhejiang University School of Medicine and can also be purchased commercially.

[0068] The LB medium formula is: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and 1 L deionized water. Tryptone, sodium chloride, and yeast extract were purchased from OXOID.

[0069] The formula of 2×YT pre-germination solution is: 16g tryptone, 10g yeast extract, 5g NaCl, and 1L deionized water.

[0070] The formula of MS solid medium is: 20g soybean powder, 20g mannitol, 20g agar. The MS solid medium used for conjugation transfer needs to be supplemented with MgCl at a final concentration of 10mM. 2 .

[0071] The formula of ISP2 medium is: 10 g malt extract, 4 g yeast extract, 4 g D-glucose, 20 g agar, 1 L deionized water, and the pH is adjusted to 7.0-7.4.

[0072] The formula of R5 medium is: sucrose 103g, glucose 10g, yeast extract 5g, casein hydrolyzate 0.1g, K 2 SO 4 0.25 g, MgCl 2 .6H 2 O 10.12g, TES 5.73g, L-proline 0.3g, KH 2 PO 4 (5%) 1 mL, CaCl 2 (5M) 4mL, NaOH (10M) 700μL, trace element storage solution 2ml, 1L deionized water. 2 PO 4 (5%), CaCl 2 (5M), NaOH (10M) and trace element storage solution need to be prepared and sterilized separately. The formula of R5 trace element storage solution is as follows: ZnCl 2 40mg, FeCl 3 6H 2 O 200 mg, CuCl 2 ·2H 2 O 10mg, MnCl 2 ·4H 2 O 10 mg, Na 2 B 4 O 7 10H 2 O10mg, (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 10mg, dilute to 1L with deionized water.

[0073] The concentrations (final concentrations) used for antibiotic selection during the E. coli culture process were as follows: abramycin 50 μg / ml, spectinomycin 100 μg / ml, kanamycin 50 μg / ml, and chloramphenicol 25 μg / ml.

[0074] The concentrations (final concentrations) used for antibiotic selection during the culture of Streptomyces were as follows: abramycin 50 μg / ml, spectinomycin 50 μg / ml, and nalidixic acid (NA) 25 μg / mL.

[0075] Preparation method of glycerol spore suspension: add 1mL 20% glycerol to the sterilized bacterial tube, completely soak one end of the sterilized cotton swab in the tube, then use the cotton swab to scrape the aerial hyphae layer on the surface of the plate and immerse the cotton swab in the bacterial tube again, stirring continuously, and wash the spores and aerial hyphae picked up by the top of the cotton swab into the glycerol. After the bacterial tube is covered and the interface between the tube cover and the tube body is sealed with a sealing film, it is placed in a vortex instrument and fully shaken for 3 to 5 minutes; then, centrifuge at room temperature at 5000rpm for 5 minutes, then discard the supernatant in the clean bench, and resuspend the spore precipitate with 1mL 20% glycerol; use a vortex instrument to shake the resuspension again for 3 to 5 minutes; centrifuge at room temperature at 5000rpm for 5 minutes, then discard the supernatant in the clean bench, and resuspend the spore precipitate with 1.5 to 2mL of 20% glycerol to obtain a glycerol spore suspension. Freeze in a -80℃ refrigerator for use.

[0076] Resuscitation of glycerol spore suspension: Take out the frozen glycerol spore suspension from the -80℃ refrigerator and temporarily place it on ice. In the clean bench, use the tip of the gun to poke ice chips the size of mung beans, and evenly spread the spore droplets on the surface of the plate. Use sealing film to seal the joints of the upper and lower plates, put them in a fresh-keeping bag, and incubate them upside down in a 30℃ constant temperature incubator for 8 to 10 days. It can be used for subsequent conjugation transfer or preparation of spore suspension.

[0077] The conjugation transfer method was carried out by the conventional method described in the Genetic Manipulation of Streptomyces: A Laboratory Manual. 1985. DA Hopwood The John Innes Foundation. The specific experimental operation of conjugation transfer is as follows:

[0078] ① In the clean bench, add 1mL of TES buffer to the sterilized 1.5mL EP tube, immerse one end of the cotton swab in the liquid, and then use this end to gently scrape the surface of the plate that has been incubated for 8 to 10 days after the glycerol spore suspension was revived. Scrape the spores on the cotton swab and then immerse the cotton swab again in the TES buffer and stir to fully dissolve the spores in the TES buffer. Scrape the plate repeatedly 3 times, collect the spores on the entire plate, and obtain a suspension containing spores. Use a vortex instrument to fully shake and mix the collected spore suspension to make the spores fall off the hyphae into the liquid; centrifuge at 3000×g for 5min at room temperature, discard the supernatant, and obtain a spore pellet; resuspend the spore pellet with 1mL TES Buffer, vortex and mix or use a pipette to mix, centrifuge at 3000×g for 5min at room temperature, discard the supernatant, and repeat this step twice to obtain cleaned spores. Take 500 μL TES buffer to resuspend the washed spores, incubate at 50°C for 10 min, and immediately rinse with cold water. Add 2×YT germination solution and 5 μL of 1 M CaCl to the incubated spore suspension in a clean bench. 2 The solution (final concentration is 5 mM) was cultured in a 37°C constant temperature shaker at 200 rpm to germinate the spores for 2 hours. The germinated spore suspension was centrifuged at 3000×g for 5 minutes at room temperature, and the supernatant was discarded to obtain the germinated spore pellet, 1 mL of liquid LB medium was added, and the germinated spore pellet was resuspended by pipetting, and then centrifuged at 3000×g for 5 minutes at room temperature, the supernatant was discarded, and 500 μL of liquid LB medium was added, and the pellet was resuspended by pipetting to obtain a spore suspension.

[0079] ② While germinating the spores, use LB liquid medium without antibiotics to adjust the OD 600 The ET12567 / pUZ8002 bacterial solution containing the recombinant plasmid was cultured to about 0.6 and then washed: the OD 600 1.5 mL of ET12567 / pUZ8002 bacterial solution containing the recombinant plasmid was cultured to about 0.6, centrifuged at 3000×g and 4°C for 5 min, discarded the supernatant, resuspended the bacteria (precipitate) with 1 mL of LB liquid culture medium, and washed three times to ensure that the antibiotics were completely washed away; finally, resuspended the bacteria (precipitate) with 500 μL of LB liquid culture medium to obtain a recombinant Escherichia coli suspension.

[0080] ③ Mix 500μL of recombinant E. coli suspension obtained in step ② and 500μL of spore suspension obtained in step ①, blow and mix, a total of 1mL of mixed solution; take 200μL of the mixed solution and spread it on MS solid plate medium containing 10mM MgCl2, invert and culture at 30℃, and cover with antibiotics after about 17h: take 250μL of sterile deionized water in an Ep tube, add 25μL of nalidixic acid (NA, 25mg / mL) and the corresponding antibiotics, mix and spread evenly. The amount of each antibiotic added is as follows: 10μL of abramycin (50mg / mL), 10μL of spectinomycin (100mg / mL); invert and culture at 30℃ for 3-5d.

[0081] Template processing method in hyphae PCR: Use a sterilized toothpick to scrape a small amount of hyphae from the monoclonal culture in a clean bench. However, considering that the monoclonal culture has too few hyphae, in general, a sterilized toothpick is used to scrape a small amount of hyphae from the monoclonal culture in a clean bench, and a 2mm thick plate is streaked on an MS solid culture medium plate covered with antibiotics. 2 After inverting and culturing for 3-5 days, scrape out 2 mm 2 The mycelia were dissolved in 10 μL DMSO (the system can be appropriately reduced or enlarged according to the amount of mycelia), placed in a 100°C water bath for 15 min, placed in a -20°C refrigerator for an ice bath for 30 min, and repeated once.

[0082] 2. Sequence synthesis

[0083] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the following fragments: conduction sgRNA fragment, stimulatory sgRNA fragment, tacDC-8Fuze-sco5087 fragment, and codon-optimized TadA8e fragment.

[0084] The nucleotide sequence of gene sco5087 is shown in SEQ ID NO.7;

[0085] The nucleotide sequence of the conductive sgRNA fragment is shown in SEQ ID NO.8, which contains the conductive sgRNA and fragments with low homology to other components to facilitate recombination.

[0086] The nucleotide sequence of the stimulating sgRNA fragment is shown in SEQ ID NO. 9. The fragment contains the stimulating sgRNA and fragments with low homology to other components to facilitate recombination.

[0087] The nucleotide sequence of the tacDC-8Fuze-sco5087 fragment is shown in SEQ ID NO.11.

[0088] The nucleotide sequence of the codon-optimized TadA8e fragment is shown in SEQ ID NO.12.

[0089] The primers are as follows, synthesized by Beijing Qingke Biotechnology Co., Ltd.:

[0090] The nucleotide sequence of the first primer SMR-F is shown in SEQ ID NO.13, and the nucleotide sequence of the second primer SMR-R is shown in SEQ ID NO.14. The nucleotide sequence of the third primer 804-F is shown in SEQ ID NO.15, and the nucleotide sequence of the fourth primer 804-R is shown in SEQ ID NO.16.

[0091] The nucleotide sequence of the fifth primer CY41-F is shown in SEQ ID NO.17, and the nucleotide sequence of the sixth primer CY41-R is shown in SEQ ID NO.18.

[0092] The nucleotide sequence of the seventh primer Push-F is shown in SEQ ID NO.19, and the nucleotide sequence of the eighth primer Push-R is shown in SEQ ID NO.20; the nucleotide sequence of the ninth primer Fire-F is shown in SEQ ID NO.21, and the nucleotide sequence of the tenth primer Fire-R is shown in SEQ ID NO.22; the nucleotide sequence of the eleventh primer Fuze-F is shown in SEQ ID NO.23, the nucleotide sequence of the twelfth primer Fuze-R is shown in SEQ ID NO.24, the nucleotide sequence of the thirteenth primer 804E-F is shown in SEQ ID NO.25, and the nucleotide sequence of the fourteenth primer 804E-R is shown in SEQ ID NO.26.

[0093] The nucleotide sequence of the fifteenth primer ABE8e-F is shown as SEQ ID NO.27, the nucleotide sequence of the sixteenth primer ABE8e-R is shown as SEQ ID NO.28, the nucleotide sequence of the seventeenth primer D10ACas9-F is shown as SEQ ID NO.29, the nucleotide sequence of the eighteenth primer H840A-R is shown as SEQ ID NO.30, the nucleotide sequence of the nineteenth primer H840A-F is shown as SEQ ID NO.31, and the nucleotide sequence of the twentieth primer tipA-R is shown as SEQ ID NO.32.

[0094] The nucleotide sequence of the 21st primer Cas-F is shown in SEQ ID NO. 33, and the nucleotide sequence of the 22nd primer Cas-R is shown in SEQ ID NO. 34. The nucleotide sequence of the 23rd primer 5087-F is shown in SEQ ID NO. 35, and the nucleotide sequence of the 24th primer 5087-R is shown in SEQ ID NO. 36.

[0095] The nucleotide sequence of the twenty-fifth primer 835 is shown as SEQ ID NO.37, and the nucleotide sequence of the twenty-sixth primer 836 is shown as SEQ ID NO.38. The nucleotide sequence of the twenty-seventh primer 837 is shown as SEQ ID NO.39, and the nucleotide sequence of the twenty-eighth primer 838 is shown as SEQ ID NO.40. The nucleotide sequence of the twenty-ninth primer 839 is shown as SEQ ID NO.41, and the nucleotide sequence of the thirtieth primer 848 is shown as SEQ ID NO.42. The nucleotide sequence of the thirty-first primer Afuze-seq-L is shown as SEQ ID NO.43, and the nucleotide sequence of the thirty-second primer Afuze-seq-Rd is shown as SEQ ID NO.44.

[0096] 3. Plasmid Construction

[0097] (1) Construction of pSOK804E-SMR plasmid

[0098] ① Use the first primer and the second primer to amplify the spectinomycin resistance gene SMR fragment, and use the third primer and the fourth primer to amplify the pSOK804E plasmid backbone.

[0099] ② The above two amplified PCR product fragments were cloned in one step using ABclonal's 2×MultiF Seamless Assembly Mix reagent, as follows: After mixing the above two amplified PCR product fragments in a 1:1 molar ratio, add an equal volume of 2×MultiF Seamless Assembly Mix (equal to the volume of the two amplified fragments), connect in a 50°C water bath for 60 minutes to obtain the recombinant product, aspirate 10μL of the recombinant product and add it to the E. coli DH5α competent cells of TOLOBIO, ice bath for 30 minutes, heat shock in a 42°C water bath for 90 seconds, add 400μL of liquid LB culture medium, culture at 37°C shaker at 220rpm for 2h, centrifuge at 5000rpm for 3min, and discard the supernatant. Resuspend the bacteria with 200μL of LB liquid culture medium, and gently spread it on the LB culture medium plate containing spectinomycin with a sterile coating stick. After the bacterial solution is absorbed, invert the plate at 37°C and culture for 24h. Single colonies were picked out for colony PCR sequencing, and the correct colonies were picked out according to the sequencing results. At this point, the construction of the pSOK804E-SMR plasmid was completed.

[0100] (2) Construction of pCRISPR-cBEST-CY41 plasmid

[0101] ① Annealing with the fifth primer CY41-F and the sixth primer CY41-R to obtain the Oligo-CY41 fragment. The annealing system includes: 10 μL of primers CY41-F and CY41-R, 10 μL of deionized water; annealing PCR program: (1) 98°C for 2 min; (2) decrease by 0.1°C every 8 s to 4°C.

[0102] ② Use restriction endonuclease NcoI to digest pCRISPR-cBEST plasmid to obtain linearized vector, wherein the enzyme digestion system includes: 10×QuickCut Green Buffer 5μL, plasmid 1μg, NcoI enzyme 1μL, FastAP 1μL, add deionized water to 50μL, and incubate at 37℃ for 2h.

[0103] ③ The above-obtained Oligo-CY41 fragment and pCRISPR-cBEST linearized vector were cloned in one step using ABclonal's 2×MultiF Seamless Assembly Mix reagent, as follows: the above-mentioned Oligo-CY41 fragment and pCRISPR-cBEST linearized vector were mixed in a 1:1 molar ratio, and an equal volume (a volume equal to the sum of the volumes of the Oligo-CY41 fragment and the pCRISPR-cBEST linearized vector) of 2×MultiF Seamless Assembly Mix was added, and the recombinant product was obtained after connection in a 50°C water bath for 60 minutes. 10 μL of the recombinant product was aspirated and added to TOLOBIO's Escherichia coli DH5α competent cells, ice-bathed for 30 minutes, heat-shocked in a 42°C water bath for 90 seconds, and 400 μL of liquid LB medium was added thereto, and the culture was shaken at 37°C at 220 rpm for 2 hours, centrifuged at 5000 rpm for 3 minutes, and the supernatant was discarded. Resuspend the bacteria in 200 μL of LB liquid medium and spread it evenly on the LB medium plate containing abramycin with a sterile spreader. After the bacterial solution is absorbed, invert the plate and culture it at 37°C for 24 hours. Pick out individual colonies for colony PCR sequencing, and pick out the correct colonies based on the sequencing results. At this point, the construction of the pCRISPR-cBEST-CY41 plasmid is complete.

[0104] The plasmid map of pCRISPR-cBEST-CY41 is as follows Figure 3A The spacer sequence (CY41) on the sgRNA targeting the sco5087 gene is shown in Figure 3B shown.

[0105] (3) Construction of pSOK804E-tacDC-8Fuze-sco5087 plasmid

[0106] ①Use the seventh primer Push-F and the eighth primer Push-R to amplify the transduction sgRNA fragment, use the ninth primer Fire-F and the tenth primer Fire-R to amplify the excitation sgRNA fragment, use the eleventh primer Fuze-F and the twelfth primer Fuze-R to amplify the tacDC-8Fuze-sco5087 fragment, and use the thirteenth primer 804E-F and the fourteenth primer 804E-R to amplify the pSOK804E-SmR plasmid. The amplification conditions are as follows: PCR amplification system includes: Gloria Nova HS2×Master Mix 25μL, upstream and downstream primers 1μL each, template 1μL, deionized water 22μL. The PCR amplification procedure was as follows: (1) 98°C, 45 s; (2) 98°C, 10 s; (3) 60°C, 20 s; (4) 72°C, 2 min 40 s; steps (2) to (4) were repeated for 35 cycles; (5) 72°C, 10 min.

[0107] ② The above four amplified PCR product fragments were cloned in one step using ABclonal's 2×MultiF SeamlessAssembly Mix reagent, as follows: After mixing the above four amplified PCR product fragments in a 1:1:1:1 molar ratio, add an equal volume of 2×MultiF SeamlessAssembly Mix (equal to the volume of the four amplified fragments), connect in a 50°C water bath for 60 minutes to obtain the recombinant product, take 10μL of the recombinant product and add it to TOLOBIO's Escherichia coli DH5α competent cells, ice bath for 30 minutes, heat shock in a 42°C water bath for 90 seconds, add 400μL of liquid LB culture medium, culture at 37°C shaker at 220rpm for 2h, centrifuge at 5000rpm for 3min, and discard the supernatant. Resuspend the bacteria with 200μL of LB liquid culture medium, and gently spread it on the LB culture medium plate containing spectinomycin with a sterile coating stick. After the bacterial solution is absorbed, invert the plate at 37°C and culture for 24h. Single colonies were picked out for colony PCR sequencing, and the correct colonies were picked out according to the sequencing results. At this point, the construction of the pSOK804E-tacDC-8Fuze-sco5087 plasmid was completed.

[0108] The plasmid map of pSOK804E-tacDC-8Fuze-sco5087 is as follows Figure 4 shown.

[0109] (4) Construction of pCRISPR-PtipA-streABE8e plasmid:

[0110] ①Use the 15th primer ABE8e-F and the 16th primer ABE8e-R to amplify the codon-optimized TadA8e fragment, use the 17th primer D10ACas9-F and the 18th primer H840A-R to amplify the C-terminally truncated D10A nCas9 fragment using the pCRISPR-aBEST plasmid as a template, and use the 19th primer H840A-F and the 20th primer tipA-R to amplify the N-terminally truncated H840A nCas9 fragment and the pCRISPR-aBEST plasmid backbone using the pCRISPR-aBEST plasmid as a template. The amplification conditions are as follows: The PCR amplification system includes: Gloria Nova HS2×Master Mix 25μL, 1μL each of upstream and downstream primers, 1μL template, and 22μL deionized water. The PCR amplification procedure was as follows: (1) 98°C, 45 s; (2) 98°C, 10 s; (3) 60°C, 20 s; (4) 72°C, 4 min 30 s; steps (2) to (4) were repeated for 35 cycles; (5) 72°C, 10 min.

[0111] ② The above three amplified PCR product fragments were cloned in one step using ABclonal's 2×MultiF SeamlessAssembly Mix reagent, as follows: After the above amplified PCR product fragments were mixed at a molar ratio of 1:1:1, an equal volume of 2×MultiF SeamlessAssembly Mix (equal to the sum of the volumes of the three amplified fragments) was added, and the recombinant product was obtained after ligation in a 50°C water bath for 60 minutes. 10 μL of the recombinant product was added to the E. coli DH5α competent cells of TOLOBIO, ice bathed for 30 minutes, heat-shocked in a 42°C water bath for 90 seconds, 400 μL of liquid LB medium was added thereto, and the cells were cultured at 37°C on a shaker at 220 rpm for 2 hours, centrifuged at 5000 rpm for 3 minutes, and the supernatant was discarded. The bacteria were resuspended in 200 μL of LB liquid medium and gently spread on the LB medium plate containing abramycin using a sterile coating stick. After the bacterial solution was absorbed, the plate was inverted and cultured at 37°C for 24 hours. Single colonies were picked out for colony PCR sequencing, and the correct colonies were picked out according to the sequencing results. At this point, the construction of the pCRISPR-PtipA-streABE8e plasmid was completed.

[0112] The plasmid map of pCRISPR-PtipA-streABE8e is as follows Figure 5 shown.

[0113] IV. Transformation and Verification of Plasmids

[0114] ① Add 1000 ng of pCRISPR-cBEST-CY41 plasmid to Escherichia coli ET12567 / pUZ8002 competent cells, place on ice for 30 minutes, heat shock at 42°C in a water bath for 90 seconds, add 400 μL of LB liquid culture medium, culture at 37°C shaker at 220 rpm for two hours, and spread on LB culture medium plates containing abramycin, kanamycin, and chloramphenicol for growth. After inverted overnight culture at 37°C, pick 8 to 12 clones and inoculate them into LB liquid culture medium containing abramycin, kanamycin, and chloramphenicol for overnight culture.

[0115] ② Add 500 ng of pSOK804E-tacDC-8Fuze-sco5087 plasmid to Escherichia coli ET12567 / pUZ8002 competent cells, place on ice for 30 minutes, heat shock at 42°C in a water bath for 90 seconds, add 400 μL of LB liquid culture medium, culture at 37°C in a shaker at 220 rpm for two hours, and spread on LB culture medium plates containing triple antibodies of spectinomycin, kanamycin, and chloramphenicol for growth. After inverted overnight culture at 37°C, pick 8 to 12 clones and inoculate them into LB liquid culture medium containing triple antibodies of spectinomycin, kanamycin, and chloramphenicol for overnight culture.

[0116] ③ Add 1000 ng of pCRISPR-PtipA-streABE8e plasmid to Escherichia coli ET12567 / pUZ8002 competent cells, place on ice for 30 minutes, heat shock at 42°C in a water bath for 90 seconds, add 400 μL of LB liquid culture medium, culture at 37°C shaker at 220 rpm for two hours, and spread on LB culture medium plates containing abramycin, kanamycin, and chloramphenicol antibodies for growth. After inverted overnight culture at 37°C, pick 8 to 12 clones and inoculate them into LB culture medium containing abramycin, kanamycin, and chloramphenicol antibodies for overnight culture.

[0117] Take each bacterial solution obtained by overnight culture in the above ① to ③ for bacterial solution PCR, and set up the respective positive control plasmids for PCR amplification as a control to verify whether each target plasmid is correctly transferred into the ET12567 / pUZ8002 competent cells.

[0118] pCRISPR-cBEST-CY41 and pCRISPR-PtipA-streABE8e plasmids were verified using the upstream and downstream primer pair Cas-F / R, and pSOK804E-tacDC-8Fuze-sco5087 was verified using the upstream and downstream primer pair 5087-F / R. The PCR system includes: bacterial solution / positive control plasmid 0.2μL, 2× PCR SuperMix 5μL, upstream and downstream primers 0.4μL each, deionized water 4μL. The PCR program is as follows: (1) 98℃, 5 minutes; (2) 94℃, 20 seconds; (3) 57℃, 20 seconds; (4) 72℃, 30 seconds; Steps (2) to (4) are repeated for 40 cycles; (5) 72℃, 5 minutes. After agarose gel electrophoresis, each band in each group is compared with the corresponding control band. The band that is consistent with the corresponding control band is the bacterial solution corresponding to it, which is the verified correct bacterial solution in this group.

[0119] Take 50 μL of the verified bacterial solution and inoculate it into 5 mL of LB liquid medium containing three antibodies of corresponding resistance, and culture at 220 rpm until OD 600 The bacterial solution can be frozen at -80℃ with 20% glycerol and reused after thawing.

[0120] 5. Construction of sco5087 knockout strain Δ5087

[0121] Prepare the OD 600 The ET12567 bacterial solution containing the pCRISPR-cBEST-CY41 plasmid of about 0.6 was conjugated with the spores of Streptomyces coelicolor M145 after germination for 2 hours. After 3 to 5 days, 12 to 24 single clones were picked and streaked on the ISP2 solid medium (selective medium) plate containing abramycin. After continuing to culture for 3 to 5 days, mycelial PCR was performed on each small block, and the small block containing the editing peak was selected by sequencing results. A small amount of mycelium of the small block containing the editing peak was picked into 200 μL sterile deionized water with a sterile toothpick for dilution, and 50 μL was taken from the dilution solution and applied to the ISP2 solid medium (non-selective medium) plate without antibiotics. After inverted culture at 37°C for 3 to 5 days, 12 to 24 single clones were picked and streaked on the MS solid medium plate with abramycin. After continuing to culture for 3 to 5 days, mycelial PCR was performed on each small block, and the small block without mixed editing peaks was selected by sequencing results. Pick a small piece of hyphae without impurities, dilute and spread it on non-selective medium, and then incubate it upside down at 37℃ for 3 to 5 days. Fig. 6A As shown, 12 to 24 clones were selected and re-inoculated in the selection medium and non-selection medium and cultured at 30°C for 3 to 5 days. The growth of each clone was observed and the clones that did not grow on the selection medium but grew on the non-selection medium were selected and inoculated into TSB liquid medium for culture for 1 day. The clones were spread on MS plates without abramycin and MS plates containing abramycin. The results of culture at 30°C for 3 to 5 days are shown as follows. Figure 6B As shown, it was found that it contains abramycin ( Figure 6B -left) did not grow on MS plates without abramycin ( Figure 6B-right) on MS plates. Mycelial PCR was performed on the selected plasmid-removed clones to further verify whether the sco5087 gene was knocked out. The sequencing results showed that the CY41 target site had been completely edited (no mixed editing peaks). The glycerol spore suspension was prepared by coating on MS plates without abramycin and continued to be cultured for 8 to 10 days to obtain the Δ5087 strain without the pCRISPR-cBEST-CY41 plasmid.

[0122] 6. Joint transfer

[0123] 6.1 Obtaining Δ5087-8Fuze-sco5087 strain

[0124] Prepare the OD 600 The ET12567 bacterial suspension containing the pSOK804E-tacDC-8Fuze-sco5087 plasmid was transferred by conjugation with the Δ5087 spores after germination for 2 hours, and a single clone was picked and streaked on a MS solid medium plate covered with spectinomycin with a diameter of about 2 mm. 2 The small pieces were inverted and cultured for 3-5 days, and then mycelial PCR was performed to verify whether the plasmid integration entered the correct site of the genome. The small pieces with correct integration were inoculated into TSB liquid culture medium for 1 day, spread on MS culture medium for 8-10 days and prepared its glycerol spore suspension to obtain the Δ5087-8Fuze-sco5087 strain.

[0125] The pSOK804E plasmid has VWB integrase. Whether the plasmid is correctly integrated into the genomic site needs further verification. There are two integration sites in Streptomyces coelicolor. It is generally believed that the integration site 2 is lethal after insertion. Therefore, both integration sites need to be verified. Verification principles and methods are as follows: Figure 7 shown.

[0126] Based on the two integration sites, we designed 6 PCR primers to verify them: Primer 839 is located upstream of integration site 1, and primer 848 is located downstream of integration site 1; Primer 835 is located upstream of integration site 2, and primer 836 is located downstream of integration site 1; Primer 837 is located upstream of the integration fragment, and primer 838 is located downstream of the integration fragment. The PCR system includes: template 0.2μL, 2× PCR SuperMix 5μL, upstream and downstream primers 0.4μL each, deionized water 4μL. PCR program is as follows: (1) 98℃, 5 minutes; (2) 94℃, 20 seconds; (3) 57℃, 20 seconds; (4) 72℃, 30 seconds; steps (2) to (4) are repeated for 35 cycles; (5) 72℃, 5 minutes. No bands larger than 2000bp can be amplified under the above PCR conditions.

[0127] After the plasmid was inserted into integration site 1, the 839 / 848 primer pair could not amplify the 800bp band corresponding to the wild type, nor could it amplify the band of the corresponding size of the plasmid (>5000bp); the 837 / 839 primer pair and the 838 / 848 primer pair could amplify the 726bp and 882bp bands that the wild type could not amplify; when the plasmid was inserted into integration site 2, the 835 / 836 primer pair could not amplify the 703bp band corresponding to the wild type, nor could it amplify the band of the corresponding size of the plasmid (>5000bp), and the 835 / 837 primer pair and the 838 / 836 primer pair could amplify the 732bp and 781bp bands that the wild type could not amplify. Figure 7 In the figure, Lane 1 represents wild-type M145, and Lanes 2 to 4 represent the insertion verification of the VWB integration site of three monoclonal clones. We can see that the bands of the primer pairs of Lanes 2 to 3 are in line with expectations, while the 839 / 849 primer pair band of Lane 4 shows the same 800bp band as the wild-type of Lane 1, which means that there may be wild-type strains mixed in, and the obtained colonies are impure and do not meet the requirements of subsequent experiments.

[0128] 6.2 A BioFuze system conjugation transfer to Streptomyces Δ5087-8 Fuze-sco5087 strain

[0129] Prepare the OD 600 The ET12567 bacterial solution containing the pCRISPR-PtipA-streABE8e plasmid was transferred by conjugation with the Δ5087-8Fuze-sco5087 spores after germination for 2 hours, and a single clone was picked and streaked on a MS solid medium plate covered with abramycin, spectinomycin and NA for about 2 mm 2 The small pieces were inverted and cultured for 3-5 days (hyphae PCR was performed to monitor the editing of Fuze sequence); the small pieces were then inoculated into TSB liquid culture medium for 1 day and then into R5 culture medium. Mycelium PCR and real-time fluorescence quantitative PCR were performed at different times during the culture process to monitor the editing of Fuze sequence and the transcription level of sco5087 gene. Among them, the PCR system used to monitor the editing of Fuze sequence by streABE8e includes: template 1μL, 2× PCR SuperMix 25μL, Afuze-seq-L / Afuze-seq-R 1μL each, deionized water 22μL. PCR program is as follows: (1) 94℃, 4 minutes; (2) 94℃, 20 seconds; (3) 57℃, 20 seconds; (4) 72℃, 20 seconds; Step (2)-Step (4) repeated 30 cycles; (5) 72℃, 5 minutes. The PCR stock solution was directly Sanger sequenced to determine the editing status of the Fuze sequence.

[0130] The sequencing results at different times after the ABioFuze system was transferred to the Streptomyces Δ5087 strain are shown in the figure. Figure 8 As shown in Figure 1, it can be seen that the 8-length Fuze sequences were completely edited by streABE8e sequentially, and finally the target site located in the promoter was completely edited at day 15. The results of real-time quantitative PCR analysis are shown in Figure 1. Fig. 9 As shown, it can be seen that the transcription level of sco5087 increased significantly after 15 days (P<0.0001), indicating that the designed actinomycete rhodopsin synthesis gene expression regulation system can achieve the timed expression regulation of sco5087, a key gene for actinomycete rhodopsin synthesis in Streptomyces. This proves that the purpose of regulating Streptomyces gene expression can be achieved by pre-programming the length of the regulatory element sequence.

[0131] In the above embodiments, an example of turning on the expression of the target gene is given. In fact, replacing the excitation target with the suppression target can achieve the purpose of turning off the expression of the target gene.

[0132] Thus, the present invention constructs an actinomycete gene expression regulation system (Actinomycete Biofuze, referred to as ABioFuze) by designing and modifying the gene promoter of the model actinomycete (Streptomyces coelicolor), optimizing codons, and constructing expression vectors, etc., and sequentially edits several targets on the pre-designed tandemly arranged DNA sequence through the single-base editor streABE8e, and finally targets and modifies the promoter-35box sequence of the Streptomyces gene, so as to close or open the promoter overlapped by the last target, and achieve the purpose of delayed regulation of the expression of the target gene. ABiofuze of the present invention can control the expression time of genes such as the antibiotic synthesis pathway of actinomycetes such as Streptomyces by presetting the number (length) of tandem conduction sequences, and the number of targets (length of conduction sequences) can be adjusted according to actual requirements.

[0133] The target gene sco5087 in the example of the present invention can be replaced by other reporter genes. Similarly, it is also feasible to select functional genes or regulatory factors (such as natural product anabolism, antibiotic anabolism pathway genes) as target genes. Therefore, the application of the above-mentioned gene expression regulatory elements or systems includes but is not limited to: pre-programming of different metabolite pathway synthesis genes for actinomycetes such as Streptomyces for sequential expression; and gene expression control for industrial actinomycete strains for natural product and antibiotic synthesis.

Claims

1. A dynamic regulatory switch element for actinomycete gene expression, comprising a regulatory element for turning on downstream gene expression or a regulatory element for turning off downstream gene expression, wherein: The overall sequence composition of the regulatory element for turning on downstream gene expression is as follows: 5'-TacDD / TacDC promoter sequence-stimulation target sequence-tandem DNA sequence basic unit × n-NBB-TGG-3'; The overall sequence composition of the regulatory element for shutting down downstream gene expression is as follows: 5'-KasOpCD promoter sequence-suppression target sequence-tandem DNA sequence basic unit × n-NBB-TGG-3'; Wherein, N represents any base, B represents one of the bases G, T, and C, and n represents the number of basic units, n = 1, 2, 3, ...; Among them, the TacDD promoter sequence is shown as SEQ ID NO.1, the TacDC promoter sequence is shown as SEQ ID NO.2; the KasOpCD promoter sequence is shown as SEQ ID NO.3; the tandem DNA sequence basic unit is shown as SEQ ID NO.4; the excitation target sequence is shown as SEQ ID NO.5, and the suppression target sequence is shown as SEQ ID NO.

6.

2. The dynamic regulation switch element for actinomycete gene expression according to claim 1, characterized in that: {Tandem DNA sequence basic unit × n-NBB-TGG} includes 1 initial conduction sequence and n-1 conduction sequences in the initial state, wherein the composition of the initial conduction sequence is the tandem DNA sequence basic unit - NBB-TGG, and the composition of the conduction sequence is the tandem DNA sequence basic unit - NBB-TAA, there is an overlapping region between the 5' end of the target of the initial conduction sequence and the 3' end of the target of the conduction sequence, there is an overlapping region between the adjacent 5' ends and 3' ends of the targets of any two conduction sequences, the target of the initial conduction sequence and the target of the conduction sequence share a conduction sgRNA, and the nucleotide sequence of the conduction sgRNA is shown in SEQ ID NO.

8.

3. The dynamic regulation switch element for actinomycete gene expression according to claim 1, characterized in that: The sgRNA used for the excitation target / suppression target is called excitation / suppression sgRNA, the nucleotide sequence of the excitation sgRNA is shown in SEQ ID NO.9, and the nucleotide sequence of the suppression sgRNA is shown in SEQ ID NO.

10.

4. A dynamic regulation system for actinomycete gene expression, comprising: The single-base editing enzyme expression plasmid pCRISPR-PtipA-streABE8e plasmid and the pSOK804E-Fuze plasmid containing the Fuze-like sequence; The pCRISPR-streABE8e plasmid is obtained by the following method: replacing ABE7.10 of pCRISPR-aBEST with codon-optimized TadA8e to obtain pCRISPR-PtipA-ABE8e-nCas9; and then performing a point mutation D10A on nCas9 of pCRISPR-PtipA-ABE8e-nCas9 to obtain the pCRISPR-PtipA-streABE8e plasmid; The pSOK804E-Fuze plasmid is obtained by the following method: cloning the dynamic regulatory switch element, actinomycete target gene, conduction sgRNA and stimulation / suppression sgRNA as described in any one of claims 1 to 3 into the pSOK804E plasmid to obtain the pSOK804E-Fuze plasmid.

5. The dynamic control system according to claim 4, characterized in that: The target gene includes a functional gene or a regulatory factor.

6. The dynamic control system according to claim 4, characterized in that: The target gene is sco5087.

7. A method for dynamically regulating gene expression of actinomycetes, comprising: (1) Designing a dynamic regulatory switch element for actinomycete gene expression according to any one of claims 1 to 3, cloning the dynamic regulatory switch element, the actinomycete target gene, the conducting sgRNA and the stimulating / repressing sgRNA into a pSOK804E plasmid, thereby obtaining a pSOK804E-Fuze plasmid; (2) Replacing ABE7.10 of pCRISPR-aBEST with codon-optimized TadA8e to obtain pCRISPR-PtipA-ABE8e-nCas9; then performing a point mutation D10A on nCas9 of pCRISPR-PtipA-ABE8e-nCas9 to obtain pCRISPR-PtipA-streABE8e plasmid; (3) The pSOK804E-Fuze plasmid and the pCRISPR-PtipA-streABE8e plasmid are sequentially transferred into the target actinomycetes by conjugation transfer or preparation of actinomycete protoplasts.

8. The method for dynamic regulation of actinomycete gene expression according to claim 7, characterized in that: In step (3), the pSOK804E-Fuze plasmid and the pCRISPR-PtipA-streABE8e plasmid are sequentially transferred into the target actinomycetes by conjugation transfer as follows: the Escherichia coli ET12567 bacterial solution containing the pSOK804E-Fuze plasmid is conjugated with the pre-germinated target actinomycete spores to form integrated strain spores; the Escherichia coli ET12567 bacterial solution containing the PCRISPR-PtipA-streABE8e plasmid is conjugated with the integrated strain spores to transfer the dynamic regulation system into the target actinomycetes.

9. The application of the dynamic control switch element as described in any one of claims 1 to 3 includes, but is not limited to: being used for pre-programming the temporal expression of genes for synthesis of different metabolite pathways of actinomycetes; or being used for controlling the gene expression of actinomycete strains in the natural product and antibiotic synthesis industries.

10. The application of the dynamic control system as claimed in any one of claims 4 to 6, including but not limited to: pre-programming the temporal expression of genes synthesizing different metabolite pathways of actinomycetes; or controlling the gene expression of actinomycete strains in the natural product and antibiotic synthesis industries.

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