CN120683079A

CN120683079A - A tool for deleting large DNA fragments in Streptomyces, its recombinant plasmid and its application

Info

Publication number: CN120683079A
Application number: CN202510597499.7A
Authority: CN
Inventors: 童垚俊; 罗晶; 谢苡恩
Original assignee: Shanghai Jiao Tong University
Current assignee: Shanghai Jiao Tong University
Priority date: 2025-05-09
Filing date: 2025-05-09
Publication date: 2025-09-23

Abstract

The invention discloses a tool for deleting large segments of streptomycete DNA, a recombinant plasmid and application thereof, wherein the tool for deleting large segments of streptomycete DNA comprises TnpB nuclease, guide RNA and a DNA large segment deleting box. The DNA large fragment deletion box comprises an upstream homology arm of the target DNA large fragment and a downstream homology arm of the target DNA large fragment, wherein the lengths of the upstream homology sequences and the downstream homology sequences of the target DNA large fragment are respectively 1.5-10kb. The invention also discloses a recombinant plasmid comprising the streptomycete DNA large fragment deleting tool, application of the streptomycete DNA large fragment deleting tool or the recombinant plasmid in knocking out the streptomycete DNA large fragment, and a knocking-out method of the streptomycete DNA large fragment. The invention combines the homologous recombination system with the high-efficiency TnpB mini gene editing system, can realize the accurate and high-efficiency editing of the large segment of the streptomycete DNA, and has great popularization and application values.

Description

Streptomyces DNA large fragment deleting tool, recombinant plasmid and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a streptomycete DNA large fragment deleting tool, a recombinant plasmid and application thereof.

Background

The large fragment editing of the genome DNA of the microorganism is an important technical means for simplifying the genome, optimizing the metabolic pathway and researching the gene. Streptomyces (Streptomyces) is the largest genus among actinomycetes, and is considered to be a very development-valuable group because it produces a large number of valuable active secondary metabolites and also contains abundant silent biosynthetic gene clusters in its genome. However, streptomyces has a large genome (8-10 Mb) and a high GC content (generally GC content of more than 70%), and compared with other microorganisms, genetic operations such as gene editing are difficult, genetic operation means are limited, and particularly effective tools are lacking when large genome fragment editing is performed.

TnpB protein is a programmable nuclease discovered in transposon systems in recent years, is evolutionarily likely to be an ancestor of Cas12 protein, can be guided by long non-coding RNA of about 231nt to cleave DNA sequences near the 5' end of TTGAT, and has a volume of only about 1/3 (about 400 amino acids) of Cas protein, and is attracting attention of researchers at home and abroad. To date, tnpB nucleases have been successfully used for endogenous gene editing in human cells, mouse embryos, monocots, dicots, and the like. Researchers have also performed extensive and systematic mining and research of TnpB nucleases widely distributed in organisms to identify a variety of TnpB with targeted editing activity. However, as a whole, tnpB has a problem of low editing efficiency compared with Cas9, and more importantly, the existing small-sized editor has not been reported for in vivo gene editing of streptomycete.

Patent CN113528408B discloses a method for deleting large fragments of high-efficiency genome based on CRISPR-nCas3 system and application, which are mainly applied to zymomonas mobilis and similar cells, wherein the zymomonas mobilis and similar cells have significant differences in physiological characteristics and the like, the zymomonas mobilis and similar cells are gram-negative bacteria, the genome is smaller (2-3 Mbp), the streptomyces is gram-positive bacteria, the genome is larger (8-12 Mbp), the GC content is up to more than 70%, and the streptomyces mobilis has multicopy genes and repeated sequences. The application of the CRISPR-nCas-based large fragment deletion technology to streptomycete genome editing has significant challenges, and multiple obstacles such as delivery efficiency, genome complexity, genetic tool compatibility and the like need to be overcome, so that the application difficulty of the CRISPR-nCas-based large fragment deletion technology in streptomycete is high.

Disclosure of Invention

Aiming at the current situation that a large genome segment editing tool is not available in streptomyces, the invention deeply analyzes a small-volume programmable TnpB nuclease to realize a core element for gene editing and an action mechanism thereof, and develops a high-efficiency large segment gene editing tool suitable for the streptomyces by utilizing a TnpB system and a homologous recombination system, thereby realizing high-efficiency editing of a large segment gene region of the streptomyces, providing a set of powerful tool for developing basic research and application research in the streptomyces, and promoting development of metabolic engineering, systematic biology and synthetic biology.

In order to solve the technical problems, the invention aims to provide a tool for deleting large fragments of streptomycete DNA, and recombinant plasmids and application thereof.

The aim of the invention is realized by the following technical scheme:

in a first aspect, the invention provides a streptomycete DNA large fragment deletion tool based on TnpB system, comprising TnpB nuclease, guide RNA and DNA large fragment deletion cassette.

As some specific embodiments of the invention, the DNA large fragment deletion cassette sequentially comprises the following core elements of an upstream homology arm of a target DNA large fragment and a downstream homology arm of the target DNA large fragment according to the direction of editing the target DNA large fragment, and homologous recombination repair templates are provided when the homologous sequences on the upper side and the downstream side of the target DNA large fragment are used for deleting the DNA large fragment, wherein the lengths of the homologous sequences on the upper side and the downstream side of the target DNA large fragment are respectively 1.5-10kb.

As some embodiments of the invention, the guide RNA comprises an RNA backbone, a targeting segment, and a Hepatitis Delta Virus (HDV) ribozyme.

As some specific embodiments of the invention, the nucleotide sequence of the RNA skeleton is shown as SEQ ID NO.3, the targeting segment is positioned at the 3 'end of the RNA skeleton and is a nucleic acid sequence with the length of 12-40nt after a TAM sequence (5' TTGAT) on a large fragment of a targeted gene, and the nucleotide sequence of the Hepatitis Delta Virus (HDV) ribozyme is shown as SEQ ID NO.5 and is used for stabilizing the structure of the RNA skeleton-gene targeting segment.

As some specific embodiments of the invention, the TnpB nuclease is TnpB nuclease which can be expressed in streptomyces through codon optimization, the DNA similarity with TnpB of a wild type Deinococcus radiodurans ISDra2 source is 79.74%, the amino acid sequence of the TnpB nuclease is shown as SEQ ID NO.1, and the nucleotide sequence of the TnpB nuclease is shown as SEQ ID NO. 2.

In a second aspect, the present invention provides a recombinant plasmid comprising a deletion tool for large fragments of Streptomyces DNA as described in one of the above. The recombinant plasmid is a streptomycete DNA large fragment deletion plasmid containing an apramycin resistance screening marker.

As some specific embodiments of the invention, the recombinant plasmid is constructed by respectively inserting TnpB nuclease, guide RNA and a DNA large fragment deletion cassette on the basis of an escherichia coli-streptomyces shuttle plasmid.

As some specific embodiments of the present invention, the construction method of the recombinant plasmid comprises:

S1, respectively replacing a guide RNA and a carrier RNA with TnpB nuclease to obtain plasmids pTnpB-reRNA by using Cas9 and sgRNA fragments on a streptomycete-escherichia coli shuttle plasmid vector pCRISPR-Cas 9;

s2, carrying out enzyme digestion on the plasmid pTnpB-reRNA by SphI endonuclease to obtain linearization pTnpB-reRNA;

s3, performing seamless cloning and assembly on the DNA large fragment deletion cassette and the linearization plasmid vector pTnpB-reRNA to obtain plasmid pSTAGE-BGC.

In a third aspect, the present invention provides a use of a deletion tool or recombinant plasmid for large segments of Streptomyces DNA as described in any one of the above, in knockout of large segments of Streptomyces DNA.

In a fourth aspect, the invention provides a method for knocking out a large segment of Streptomyces DNA, comprising the following steps:

A1, under the non-induction condition, transforming the recombinant plasmid of any one of the above into a target host, realizing the deletion of the large DNA fragment by the homologous exchange of the homologous arm sequence of the large DNA fragment deletion box and the homologous sequence at the upstream and downstream of the target large DNA fragment, and obtaining a transformant carrying the large DNA fragment deletion plasmid by screening antibiotics with plasmid related resistance;

a2, streaking and inoculating the transformant obtained in the step A1 to a culture medium plate containing plasmid resistance antibiotics and promoter inducers, and culturing at constant temperature until the single clone is visible;

A3, randomly selecting the monoclonal in the step A2, and performing colony PCR verification to obtain the traceless DNA large fragment knockout strain.

The invention combines TnpB nuclease with homologous recombination repair system, and can delete gene cluster of more than 10kb by single operation. Compared with Cas9 and the like, the miniaturized structure (400 aa) of the target sequence is more suitable for the transformation bottleneck of streptomyces, and the reRNA-dependent targeting mechanism can accurately identify the TAM sequence with high AT of the streptomyces genome, so that the off-target risk of a CRISPR system is avoided.

Compared with the prior art, the invention has the following beneficial effects:

1) Combining the homologous recombination system with a high-efficiency TnpB mini gene editing system, the system develops a novel large fragment deleting tool which is applicable to streptomycete DNA;

2) The tool for deleting the large segment of the streptomycete DNA can realize the accurate and efficient editing of the large segment of the streptomycete DNA, and has great popularization and application values;

3) The deletion tool for the large segment of the streptomycete DNA can realize the efficient editing of the large segment gene region of the streptomycete, provides a powerful tool for developing basic research and application research in the streptomycete, and promotes the development of metabolic engineering, system biology and synthetic biology;

4) The invention focuses on combining TnpB nuclease with a homologous recombination repair system for the first time, can delete more than 10kb gene clusters by single operation, is more suitable for the transformation bottleneck of streptomyces compared with Cas9 and the like, and can accurately identify the TAM sequence of high AT of the streptomyces genome by a reRNA-dependent targeting mechanism, thereby avoiding the off-target risk of a CRISPR system.

Drawings

Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:

FIG. 1 is a map of pSTAGE-BGC plasmid constructed in example 1;

FIG. 2 is a graph showing the results of pigment secretion of randomly selected monoclonal editing strains containing plasmids pSTAGE-BGC-1.0kb, pSTAGE-BGC-1.5kb, pSTAGE-BGC-2.0kb and pSTAGE-BGC-2.5kb in example 3 after culturing for 14 days;

FIG. 3 is a schematic diagram of the theoretical genome of a strain from which the actinorhodin synthesis gene cluster of example 3 was successfully deleted;

FIG. 4 is a diagram showing the result of DNA electrophoresis of a part of the monoclonal DNA deleted from the actinorhodin synthesis gene cluster by pSTAGE-BGC-1.0 in example 3;

FIG. 5 is a diagram showing the result of DNA electrophoresis of a part of the monoclonal DNA deleted from the actinorhodin synthesis gene cluster by pSTAGE-BGC-1.5kb in example 3;

FIG. 6 is a graph showing the results of Sanger sequencing of a portion of a large fragment of a target gene in S.ceoliolor M145, a Streptomyces species model strain successfully knocked out using pSTAGE-BGC-1.5kb in example 3.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.

The embodiment of the invention uses streptomycete model strain Streptomyces coelicolorM as a target strain, and the genome sequence number of the strain is GeneBank: GCA_008931305.1. According to the embodiment of the invention, a key gene SCO5087 (actI) for synthesizing actinorhodin in the strain is taken as an endogenous gene target, a actinorhodin synthesis gene cluster is deleted, and the editing efficiency of the invention is tested.

EXAMPLE 1 construction of Streptomyces DNA Large fragment deletion tool

1.1 Plasmid design and construction

According to the codon preference of streptomyces, the TnpB nuclease from Deinococcus radiodurans ISDra is subjected to codon optimization, the DNA similarity of the optimized TnpB nuclease and the TnpB nuclease from Deinococcus radiodurans ISDra2 wild type is 79.74%, the amino acid sequence is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2.

The guide RNA is designated reRNA, and comprises the following core elements, namely an RNA framework (with a sequence shown as SEQ ID NO. 3), a gene targeting segment (with a sequence shown as SEQ ID NO.4 when the SCO5087 gene is taken as a target point) and a Hepatitis Delta Virus (HDV) ribozyme (with a sequence shown as SEQ ID NO. 5) in sequence according to the direction from the 5 'end to the 3' end. The above gene fragments were synthesized by Kirschner Biotech Co., ltd.

Plasmid pTnpB-reRNA was obtained by replacing the Cas9 and sgRNA fragments on the Streptomyces coli shuttle plasmid vector pCRISPR-Cas9 (https:// doi.org/10.1021/acslynbio.5b00038) with the codon optimized TnpB nuclease and reRNA-ZH, respectively.

The plasmid pTnpB-reRNA was then digested with SphI endonuclease to obtain linearization pTnpB-reRNA. And then, the gene knockout boxes formed by the upper and the lower homologous arms of the target gene are respectively assembled with linearization plasmid vectors pTnpB-reRNA in a seamless cloning manner (the kit is purchased from Vazyme and ClonExpress Ultra One Step Cloning Kit), so as to obtain a plasmid pSTAGE-BGC (the plasmid map is shown in figure 1).

The invention designs and constructs plasmids pSTAGE-BGC with upstream and downstream homology arms of about 1.0kb, 1.5kb, 2.0kb and 2.5kb, which are respectively named pSTAGE-BGC-1.0kb, pSTAGE-BGC-1.5kb, pSTAGE-BGC-2.0kb and pSTAGE-BGC-2.5kb, by taking a key gene SCO5087 (actI) synthesized by actinorgasm as an endogenous gene target point, and is used for knocking out actinorgasm synthesis gene clusters (17.4 kb). Wherein the upstream homology arm sequence with the length of about 1.0kb is shown as SEQ ID NO.6, the downstream homology arm sequence with the length of about 1.0kb is shown as SEQ ID NO.7, the upstream homology arm sequence with the length of about 1.5kb is shown as SEQ ID NO.8, the downstream homology arm sequence with the length of about 1.5kb is shown as SEQ ID NO.9, the upstream homology arm sequence with the length of about 2.0kb is shown as SEQ ID NO.10, the downstream homology arm sequence with the length of about 2.0kb is shown as SEQ ID NO.11, the upstream homology arm sequence with the length of about 2.5kb is shown as SEQ ID NO.12, and the downstream homology arm sequence with the length of about 2.5kb is shown as SEQ ID NO.13. The constructed plasmids were all Sanger sequenced to ensure complete correctness.

Example 2 application of Streptomyces high-efficient ultra Mini editing System

2.1 Conversion

The plasmid of interest constructed in example 1 was transferred into competent cells of E.coli ET12567/pUZ8002 (https:// doi. Org/10.1016/0378-1119 (92) 90549-5) for plasmid amplification, as follows:

200ng of plasmid was added to 100. Mu.L of thawed self-made competent cells E.coli ET12567/pUZ8002, gently mixed with a sterile coating rod, left on ice for 30 minutes, left on a water bath at 42℃for 45 seconds, immediately left on ice for 2-3 minutes, then added with an antibiotic-free LB liquid medium, incubated at 200rpm for 1 hour at 37℃and centrifuged at 5000rpm for 5 minutes to discard 900. Mu.L of supernatant, the bacterial body was resuspended in the remaining medium, added to LB solid plates containing kanamycin (25. Mu.g/mL), chloramphenicol (12.5. Mu.g/mL) and apramycin (50. Mu.g/mL) and gently spread with a sterile coating rod, after overnight incubation at 37℃a single clone was picked up to 20mL of LB liquid medium containing 25. Mu.g/mL, 12.5. Mu.g/mL chloramphenicol and 50. Mu.g/mL apramycin, and centrifuged at 5000rpm for 5 minutes at about 0.4 OD600, 20mL of antibiotic-free LB liquid medium was added, centrifuged at 5000rpm for 5 minutes and the supernatant was repeated two times, and the suspension was finally used for 2mL of liquid medium.

2.2, Binding transfer and resistance screening

Under non-induction conditions, the plasmid from E.coli of step 2.1 was transferred into Streptomyces coelicolor M145 (i.e., streptomyces coelicolor M145) by the following procedure:

Taking Streptomyces spores collected in advance, centrifuging at 5000rpm for 5 minutes, discarding supernatant, resuspending thalli by using 2mL of 2 XYT liquid culture medium, placing a centrifuge tube filled with spores in a 50 ℃ water bath kettle for heat shock for 10 minutes, then pre-germinating for 30 minutes in a 30 ℃ shaker at 200rpm, taking 500 mu L of E.coli bacterial liquid collected in the step 2.1 in a 1.5mL centrifuge tube containing 200 mu L of Streptomyces spore suspension, uniformly mixing, taking 200 mu L of mixed liquid, uniformly coating on an MS flat plate, pouring the MS flat plate into a 30 ℃ incubator for culture, covering the surface of the culture medium with 1mg/mL of apramycin and 1mg/mL of nalidixic acid after 18 hours, and continuously pouring the MS flat plate into the 30 ℃ incubator until a binder grows (about 5 days) after the surface is dried. At this time, a binder was obtained which was successfully edited.

Example 3 evaluation of Gene editing efficiency

Randomly picking the monoclonal obtained in the step 2.2, streaking and inoculating the monoclonal onto ISP2 solid medium with plasmid resistance antibiotics (50 mug/mL apramycin, 100 mug/mL nalidixic acid) and 0.5 mug/mL thiostrepton, inverting, observing phenotype change after culturing at a constant temperature of 30 ℃ for 14 days, and evaluating the editing efficiency of different DNA large fragment knockout systems. The results are shown in FIG. 2, and the E.coli containing pSTAGE-BGC-1.0kb, pSTAGE-BGC-1.5kb, pSTAGE-BGC-2.0kb and pSTAGE-BGC-2.5kb plasmids are shown, respectively, from left to right in FIG. 2, and are randomly picked up as different monoclonal antibodies after combined transfer with S.coelicolor M145. If the actinorhodin synthetic gene cluster is knocked out successfully, the mutant strain cannot synthesize the rhodopsin, so that colonies are colorless or light in color. After 14 days of culture, randomly picked colonies growing in the resistant solid screening medium show colorless or lighter color with the extension of the length of the homology arm, which indicates that pSTAGE-BGC can successfully knock out the large target gene fragment in the Streptomyces mode strain S.ceoliolor M145, and the extension of the length of the homology arm can improve the efficiency of knocking out the large target gene fragment of Streptomyces.

Picking up the single clone edited by pSTAGE-BGC-1.0kb and pSTAGE-BGC-1.5kb in the step 2.2, streaking and inoculating to ISP2 solid culture medium with plasmid resistant antibiotics (50 mug/mL apramycin, 100 mug/mL nalidixic acid) and 0.5 mug/mL thiostrepton, inverting, culturing at 30 ℃ until the bacterial cells are visible, picking up a small amount of bacterial cells until the bacterial cells are in a PCR tube containing 20 mu L of DMSO, placing the bacterial cells at 100 ℃ for 15 minutes, refrigerating at-20 ℃ for 30 minutes, repeating the steps twice to fully lyse the cells to obtain cell lysate, and then adopting NEBThe High-Fidelity 2X Master Mix (cat# M0492) kit PCR amplified target site fragments, the primer design was as shown in Table 1, the PCR reaction system was as shown in Table 2, and the PCR reaction procedure was as shown in Table 3.

TABLE 1 PCR amplification primers targeting SCO5087 (actI)

Primer(s)	Sequence (5 '-3')	Sequence numbering
			check-actBGC1.5k-F	acgagctgagtcgggacatg	SEQ ID NO.14
actBGC-check-R	ctgcaacggtgtcagccggc	SEQ ID NO.15

TABLE 2PCR reaction System

System of	15μL
		ddH₂O	5.25μL
Forward primer (10. Mu.M, SCO 5087-LH-F)	0.75μL
		Reverse primer (10. Mu.M, SCO 5087-R)	0.75μL
2×PhantaFlashMasterMix	7.50μL
		Cell lysate (containing DMSO)	0.25μL

TABLE 3PCR reaction procedure

And (3) carrying out 1% agarose gel electrophoresis detection on the PCR amplification product, wherein the result is shown in fig. 4 and 5, and the randomly selected colony growing in the resistant solid screening culture medium is provided with the PCR product by PCR and the size accords with the theory, so that the monoclonal is proved to successfully knock out the large fragment of the target gene. Lane 1-8 in FIG. 4 and Lane 1-7 in FIG. 5 show randomly selected portions of the monoclonal antibodies after transfer of E.coli containing pSTAGE-BGC-1.0kb and pSTAGE-BGC-1.5kb plasmids, respectively, in combination with S.coelicolorM 145. If the target gene large fragment is successfully knocked out, the theoretical PCR band size is 1702bp (figure 3), and if the actinorhodin synthetic gene cluster is not knocked out, the corresponding PCR product cannot be detected in 1% agarose gel electrophoresis.

As shown in FIG. 4, 8 single clones in pSTAGE-BGC-1.0kb plasmid edit group failed to detect PCR products conforming to the theoretical size (1702 bp), indicating that pSTAGE-BGC-1.0kb failed to knock out the large fragment of the target gene (actinorhodin synthetic gene cluster) efficiently. As shown in FIG. 5, 5 PCR products conforming to the theoretical size (1702 bp) were detected in 5 out of 7 monoclonals of pSTAGE-BGC-1.5kb editing group, and these 5 monoclonals were successfully knocked out of the actinorhodin synthesis gene cluster, so that pSTAGE-BGC-1.5kb could be successfully knocked out of the large fragment of the target gene in S.ceoliolor M145 of Streptomyces model strain. FIG. 5 shows only the results of PCR detection of a portion of randomly selected monoclonal antibodies, and analysis of all randomly selected pSTAGE-BGC-1.5kb binding molecules shows that the number of clones of strains which successfully knock out the actinorhodin synthetic gene cluster accounts for about 50%, so that the knock-out efficiency of pSTAGE-BGC-1.5kb is 50%.

The PCR product was then recovered by purification using a Renzan plasmid purification kit (cat# DC 201) and sent to Sanger sequencing analysis, inc. of Jin Weizhi Biotechnology, su. As shown in FIG. 6, a diagram of the results of Sanger sequencing of a partial single clone of a large fragment of the target gene in pSTAGE-BGC-1.5kb knock-out Streptomyces model strain S.ceoliolor M145, only the sequencing results of mutant 1 and mutant 5 (corresponding to Lane1 and Lane5, respectively, in FIG. 5) are shown in FIG. 6, and the sequencing results indicate that the large gene fragment of the actinorhodin synthetic gene cluster of 1702bp in length was indeed successfully knocked out.

In summary, it is shown that the homology arm length is the key to affect the knockout of large fragments of target genes, and the lengths of the upstream and downstream homology arms need to be about 1.5kb or more. The results fully show that the deletion tool for the large segment of the streptomycete gene can effectively mediate deletion of the large segment of the streptomycete gene.

The media used in the examples above are as follows:

Weighing 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, dissolving in 1L of ddH ₂ O, sterilizing at 115 ℃ for 30 minutes, and storing at room temperature for later use;

MS culture medium, 10g soybean cake powder, 10g tryptone and 10g agar powder are weighed, dissolved in 500mL tap water and sterilized at 115 ℃ for 30 minutes;

ISP2 culture medium, namely weighing 10g of malt extract, 4g of yeast extract and 4g of glucose, dissolving in 1L of ddH ₂ O, regulating the pH to 7.4,115 ℃ for sterilization for 30 minutes, storing at 4 ℃ for standby, and adding 2% of agar powder if a solid culture medium is configured;

2 XYT medium 16g tryptone, 10g malt extract, 5g sodium chloride are weighed, dissolved in 1L ddH ₂ O, pH is adjusted to 7.0,115 ℃ and sterilized for 30 minutes, and stored at 4 ℃ for later use.

The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.

Claims

1. A large DNA fragment deletion tool based on the TnpB system of Streptomyces, characterized by comprising: TnpB nuclease, guide RNA and a large DNA fragment deletion cassette.

2. The Streptomyces DNA large fragment deletion tool according to claim 1 is characterized in that the DNA large fragment deletion box includes the following core elements in sequence according to the direction of editing the targeted DNA large fragment: an upstream homology arm of the targeted DNA large fragment and a downstream homology arm of the targeted DNA large fragment; the lengths of the upstream homology arm of the targeted DNA large fragment and the downstream homology arm of the targeted DNA large fragment are respectively 1.5-10 kb.

3. The Streptomyces DNA large fragment deletion tool according to claim 1, characterized in that the guide RNA comprises an RNA backbone, a targeting segment and a hepatitis D virus ribozyme.

4. The Streptomyces DNA large fragment deletion tool according to claim 3, characterized in that the nucleotide sequence of the RNA backbone is shown in SEQ ID NO.3; the targeting segment is located at the 3' end of the RNA backbone and is a nucleic acid sequence with a length of 12-40 nt after the TAM sequence on the large fragment of the targeted gene; the nucleotide sequence of the hepatitis D virus ribozyme is shown in SEQ ID NO.5.

5. The Streptomyces DNA large fragment deletion tool according to claim 1, characterized in that the TnpB nuclease is a codon-optimized TnpB nuclease that can be expressed in Streptomyces, and the amino acid sequence of the TnpB nuclease is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

6. A recombinant plasmid, characterized by comprising the Streptomyces DNA large fragment deletion tool according to any one of claims 1 to 5.

7. The recombinant plasmid according to claim 6, characterized in that the recombinant plasmid is constructed by inserting TnpB nuclease, guide RNA and DNA large fragment deletion cassette into the Escherichia coli-Streptomyces shuttle plasmid respectively.

8. The recombinant plasmid according to claim 7, wherein the construction method of the recombinant plasmid specifically comprises:

S1. Replace the Cas9 and sgRNA fragments on the Streptomyces-Escherichia coli shuttle plasmid vector pCRISPR-Cas9 with TnpB nuclease and guide RNA, respectively, to obtain the plasmid pTnpB-reRNA;

S2. Cut the plasmid pTnpB-reRNA with SphI endonuclease to obtain linearized pTnpB-reRNA;

S3. Seamlessly clone and assemble the large DNA fragment deletion cassette into the linearized plasmid vector pTnpB-reRNA to obtain the plasmid pSTAGE-BGC.

9. Use of the Streptomyces DNA large fragment deletion tool according to any one of claims 1 to 5 or the recombinant plasmid according to any one of claims 6 to 8 in knocking out Streptomyces DNA large fragments.

10. A method for knocking out a large DNA fragment of Streptomyces, comprising the following steps:

A1. Under non-inducing conditions, the recombinant plasmid described in any one of claims 6 to 8 is transformed into a target host, and transformants carrying the large DNA fragment deletion plasmid are obtained by homologous exchange of the homology arm sequences of the large DNA fragment deletion cassette with the upstream and downstream homologous sequences of the targeted large DNA fragment, and antibiotic screening for plasmid-related resistance;

A2. Streak the transformants from step A1 onto a culture medium plate containing plasmid resistance antibiotics and promoter inducer, and culture at a constant temperature until a single colony is visible;

A3. Randomly select the single clones from step A2 and perform colony PCR verification to obtain a traceless large DNA fragment knockout strain.