Chassis strain for gene cluster in-vivo traceless editing, construction method and gene cluster traceless editing method

Abstract

The invention belongs to the technical field of synthetic biology genome editing, and relates to a chassis strain for gene cluster in-vivo traceless editing, a construction method and a gene cluster traceless editing method.The chassis strain is based on escherichia coli BL21 (DE3) and integrates a CRISPR-Cas9 system and a homologous recombination system (lambda Red) of a lambda bacteriophage source; and then knocking in an inducible sgRNA expression cassette (sgT) of a targeted editing vector to construct the vector. The strain has an efficient editing capability aiming at exogenous DNA, traceless editing of a target biosynthetic gene cluster is realized, and editing plasmids can be quickly eliminated, so that iterative editing is facilitated. The method is simple, convenient and rapid to operate and high in practicability, and provides powerful technical support for metabolic engineering and synthetic biology breeding transformation of streptomyces industrial strains, activation of synthetic gene clusters of recessive natural products and excavation of novel natural products.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology genome editing, and in particular relates to a chassis strain for scarless editing of gene clusters in vivo, a construction method, and a method for scarless editing of gene clusters. Background Art

[0002] With the rapid advancement of microbial genome sequencing technology, a large number of natural product (NP) biosynthetic gene clusters (BGCs) have been discovered, representing a treasure trove of novel bioactive compounds. However, under laboratory conditions, the vast majority of BGCs remain silent in their native hosts. To unlock the potential applications of these silent gene clusters, an effective approach is to activate them in heterologous hosts with more clearly characterized genetic backgrounds and more mature genetic manipulation techniques. Accurate and efficient capture of BGCs is a key prerequisite for successful heterologous expression. Over the past few decades, numerous BGC cloning techniques have been established, significantly facilitating new drug discovery. Activating silent BGCs through heterologous expression is a primary approach for obtaining relevant potentially active natural product molecules. However, after successfully capturing these BGCs, achieving optimal heterologous expression often requires various forms of editing to adapt them to the heterologous host. However, DNA deletion, insertion, or promoter replacement in such large plasmids (often larger than 50 kb) is extremely difficult. Several approaches are currently available to address these challenges:

[0003] In vitro CRISPR / Cas9-mediated editing (ICE) technology: This method uses Cas9 to cut the recombinant plasmid containing the target product BGC to be edited in vitro, and then uses T4 DNA polymerase and T4 DNA ligase to repair the cut gene cluster, ultimately achieving editing of the target location. This method needs to be performed in vitro, so the operation difficulty is relatively low, and efficient editing of gene clusters below 50kb is achieved. However, the editing efficiency of gene clusters larger than 50kb is greatly reduced, and editing of larger gene clusters is more difficult. In addition, in vitro cutting with Cas9 also requires in vitro transcription of sgRNA, which increases the difficulty of the experiment and the cost of the experiment.

[0004] CRISPR / Cas9 combined with Gibson assembly (CGE) technology: This method also uses Cas9 to cut the recombinant plasmid containing the target product BGC to be edited in vitro, and then connects it through the Gibson isothermal one-step method. The problems of this method are similar to those of the ICE method. It is difficult to edit larger gene clusters and requires in vitro transcription of sgRNA, which is costly and complex to operate.

[0005] λRed-mediated editing in E. coli: This method utilizes the linear-loop recombination capacity (LCHR) of λRed. Editing can be achieved by simply connecting the PCR-derived fragment with the resistance gene and transforming it into a strain containing the target BGC plasmid to be edited. However, this method has obvious disadvantages. A screening marker must be added during editing, which may affect the function of the edited plasmid.

[0006] E. coli in vivo (λ Red mediated) and in vitro enzyme digestion (PacI) ligation combined technology: This method also uses the linear loop recombination capacity (LCHR) of λ Red. It only needs to connect the fragment obtained by PCR with the resistance gene and transform it into the strain containing the BGC plasmid to be edited to achieve editing. However, this method introduces a PacI restriction site on both sides of the resistance gene through PCR. After the editing is completed, the plasmid is digested with PacI in vitro to remove the resistance gene. However, after enzyme digestion, the PacI restriction site still remains at the editing position, and it is still impossible to achieve completely traceless editing.

[0007] mCRISTAR (multiple plasmid-based CRISPR / Cas9 and TAR) technology: mCRISTAR is a CRISPR / Cas9-based multi-gene editing technology combined with yeast TAR in Saccharomyces cerevisiae. This technology can simultaneously replace 6 or 8 promoters with efficiencies of 68% and 32%, respectively. mCRISTAR can be iteratively executed using two different auxotrophic markers to reconstruct any type of BGC, regardless of the complexity of its operon. However, for effective screening, the addition of auxotrophic screening markers is required, which limits the use of this method. Furthermore, yeast grows slower than E. coli, and the more complex operation is also a limiting factor.

[0008] In summary, the existing technologies for editing gene clusters have the following main drawbacks:

[0009] 1. Existing gene cluster editing technologies usually perform editing in situ and then clone, but in most hosts, they face the problems of low gene editing efficiency and difficulty in editing.

[0010] 2. Introducing mutations or replacing promoter elements while cloning gene clusters usually requires resistance genes. This is not a scarless gene cluster editing, and it is difficult to effectively edit BGCs that have already been cloned.

[0011] 3. Yeast-based TAR technology, due to the super strong recombination ability of yeast cells, is prone to problems such as fragment deletion and rearrangement when cloning or editing gene clusters for the synthesis of polyketide natural products rich in repetitive sequences. Summary of the Invention

[0012] To address the shortcomings of existing technologies, this invention utilizes CRISPR / Cas9 editing technology and the λRed recombination system to develop a highly efficient and seamless editing technique for gene clusters in Escherichia coli. This approach not only simplifies genetic manipulation and offers a fast turnaround time, but also leverages the reverse-selective effect of the CRISPR system and the efficient homologous recombination capabilities of the λRed system to achieve efficient and seamless editing of gene cluster plasmids. This provides strong technical support for the development of natural product pharmaceuticals.

[0013] The present invention adopts the following technical solutions:

[0014] In a first aspect, the present invention provides a chassis strain for scarless editing of gene clusters, which is obtained by simultaneously integrating the CRISPR-Cas9 system and the lambda phage-derived homologous recombination system (λ Red) into the genome of the recipient strain.

[0015] Furthermore, an inducible sgRNA expression cassette (sg T )system.

[0016] Preferably, the Cas9 gene is Cas9 of Streptococcus pyogenes.

[0017] Preferably, the recipient strain is Escherichia coli.

[0018] Furthermore, the recipient strain is the Escherichia coli BL21 (DE3) strain.

[0019] Preferably, the recA gene is knocked out from the genome of E. coli. Knocking out the recA gene is beneficial for improving the efficiency of λ Red-based recombination.

[0020] A second aspect of the present invention provides a method for constructing a chassis strain for scarless editing of gene clusters, characterized by comprising the following steps:

[0021] S-1. The flanking sequences of the E. coli SS9 locus (homologous arms, HA), the Cas9 gene, and the λRed system were constructed into the vector pTargetF-cat to obtain the pTarget-Cas9+λRed / HA plasmid;

[0022] S-2. Using the E. coli dual-plasmid editing system (pCas, editing vector pTarget-Cas9 + λRed / HA plasmid), the Cas9 gene and λRed system were knocked into the E. coli genome;

[0023] S-3. Based on the transformed E. coli obtained in step S-2, the recA gene in the genome of E. coli was further knocked out, and sg was knocked in at the knockout site. T system, that is, a chassis strain for scarless editing of gene clusters is obtained.

[0024] Preferably, the operation of step S-1 is as follows: first, a pTarget-HA plasmid containing sequences on both sides of the Escherichia coli SS9 site (homologous arms, HA) is constructed, sgRNA is amplified from pTargetF-cat by primers ins-gRNA F / gRNA R, HA up fragment is amplified using ins-HA up F / R as primers and BL21 (DE3) as template, HA down fragment is amplified using ins-HA down F / R as primers and BL21 (DE3) as template, the three obtained fragments sgRNA, HA up, and HA down are connected with the enzyme-linearized vector pTargetF-cat to obtain the plasmid pTarget-HA, and then the fragments of the Cas9 gene and the λRed system are recombinantly connected with the plasmid pTarget-HA to obtain the pTarget-Cas9+λRed / HA plasmid.

[0025] Preferably, the operation of step S-2 is as follows:

[0026] The pCas of the E. coli dual-plasmid editing system (a plasmid transformation with the Cas9 gene and the λRed system) was electroporated into BL21(DE3) and BL21(DE3) / pCas competent cells were prepared;

[0027] The editing vector pTarget-Cas9+λRed / HA plasmid was electroporated into BL21(DE3) / pCas competent cells, plated onto kanamycin and spectinomycin double-resistant LB plates, and cultured overnight; the grown colonies were verified, and the correct colonies were inoculated into LB test tubes for overnight culture, kanamycin was added, and IPTG was added to induce the expression of sgRNA to cut the editing plasmid, thereby removing the editing vector pTarget-Cas9+λRed / HA; the plasmid pCas (containing a temperature-sensitive promoter) was removed by passage at 37°C to obtain the modified Escherichia coli (BL23) with the knock-in Cas9 gene and λRed system.

[0028] The third aspect of the present invention provides a method for scarless editing of a gene cluster based on the above-mentioned chassis strain.

[0029] The present invention is based on Escherichia coli BL21 (DE3), integrates the CRISPR-Cas system and the λRed system, and knocks out the recA gene while integrating the sg T The BL23 strain was constructed using a system. This strain possesses the ability to efficiently edit exogenous DNA, enabling scarless editing of target biosynthetic gene clusters and rapid elimination of the editing plasmid, facilitating iterative editing. This method is simple, rapid, and highly practical, providing strong technical support for metabolic engineering and synthetic biology breeding of industrial Streptomyces strains, as well as for activating cryptic natural product synthesis gene clusters and discovering new natural products.

[0030] By implementing the above technical solution, the present invention has the following advantages:

[0031] 1. The present invention integrates CRISPR / Cas9 and λRed systems into the genome of the recipient strain to construct a chassis strain, which achieves efficient and traceless editing of exogenous plasmids containing the target natural product BGC. It is more stable and the editing efficiency of single genes in the gene cluster is close to 100%.

[0032] 2. The present invention further knocks in sg on the basis of CRISPR / Cas9 and λRed system T After a round of gene cluster editing is completed, the system can be induced by IPTG to cut and eliminate the targeted plasmid, facilitating the next round of iterative editing.

[0033] 3. The present invention further knocks out the recA gene in the genome based on the CRISPR / Cas9 and λRed systems, which is beneficial to improving the recombination efficiency based on λRed.

[0034] 4. The present invention further utilizes E. coli, which is simple, fast, and has strong iterative capabilities. Previously, editing of biosynthetic gene clusters was often performed in yeast, which is relatively complex to operate, grows relatively slowly, and is difficult to obtain edited plasmids. Using E. coli can avoid these shortcomings. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1Schematic diagram of the editing process of the strain BL23 of the present invention for the natural product biosynthesis gene cluster; in the figure, ① the plasmid to be edited is introduced into the BL23 competent cell and prepared into an electroporation competent cell; ② the editing plasmid containing the sgRNA targeting the DNA to be edited and the repair template is introduced into the competent cell in step ①, and colonies are grown the next day to verify whether editing has occurred by PCR; ③ the positive colonies are transferred to a 4 mL liquid LB test tube and IPTG is added to induce sg T ④ Obtain a strain that has eliminated the editing plasmid, so that the next round of editing experiments can be carried out.

[0036] Figure 2 Schematic diagram of the screening results of Escherichia coli hosts suitable for efficient transformation of large plasmids in the present invention;

[0037] Figure 3 This is a schematic diagram of the construction process of the strain BL23 of the present invention;

[0038] Figure 4 To demonstrate the editing efficiency of the pCL01-omil plasmid carrying 5-ketomilbemycin BGC using the method of the present invention;

[0039] Figure 5 Sequencing results demonstrating precise editing of the pCL01-omil gene cluster using the method of this invention. Figures include: (A) Knockout verification of the milE gene within the gene cluster. (B) Sequencing verification of the milF gene insertion. (C) Sequencing verification of the replacement of the milR promoter with kasOp*.

[0040] Figure 6Functional verification of the 5-ketomilbemycin BGC after editing using the method of the present invention; Figure: (A) HPLC analysis of fermentation products from heterologous expression of the edited vector pCL01-omil::milF in the Streptomyces avermitilis MSB01Δave chassis, and overexpression of the edited vector pCL01-omil::milF in Streptomyces bingchengensis SIPI-054. Standard: Milbemycin A3 and A4 standards; SIPI-054: Starting strain of Streptomyces bingchengensis; SIPI-Mil: Multi-copy engineered strain after the pCL01-omil::milF plasmid was introduced into SIPI-054; MSB01Δave: Streptomyces avermitilis chassis strain lacking the avermectin biosynthetic gene cluster; MSB::mil: Heterologous expression strain with the pCL01-omil::milF plasmid introduced into the MSB01Δave chassis. (B) Milbemycin fermentation yields in a heterologous expression strain of Streptomyces avermitilis and a modified strain of Streptomyces bingchengensis expressing multiple copies of the milbemycin biosynthesis gene cluster. Heterologous milbemycin synthesis was successfully achieved in Streptomyces avermitilis MSB01Δave. Overexpression in the SIPI-054 strain increased milbemycin yield from 2000 mg / L to 4500 mg / L, more than doubling the yield. DETAILED DESCRIPTION

[0041] The following is a clear and complete description of the technical solutions in the embodiments of the present invention with reference to the accompanying drawings. The embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0042] In the following examples, using E. coli BL21 (DE3) as the chassis, CRISPR / Cas9 editing technology combined with the λRed recombination system was used to create iCASRED, an editing technology for efficient and seamless gene cluster modification in E. coli. The general process is as follows: First, the Cas9 gene and the α, β, and γ genes encoding the λRed system, as well as the sgRNA for plasmid elimination, were inserted into the Cas9 gene. T Modules were knocked into the BL21 (DE3) strain and the recA gene was deleted to obtain the BL23 strain ( Figure 1 ). Next, the plasmid containing the target product gene cluster to be edited is introduced into BL23 and prepared into competent cells. Then, the editing plasmid containing the gRNA targeting the gene cluster editing site and the repair homology arm is introduced into the above competent cells. Subsequently, the arabinose-induced editing system is added to perform targeted cutting and repair of the BGC target site, thereby achieving efficient editing of BGC. After successful editing, IPTG is added to induce sg TModule expression, thereby cutting and eliminating the editing plasmid, so that different editing plasmids can be introduced for the next round of editing ( Figure 1 ).

[0043] Example 1

[0044] Construction of chassis strains for scarless editing of gene clusters

[0045] In this example, the CRISPR / Cas9 and λRed systems were co-implanted into the BL21(DE3) genome to generate the BL22 strain. This approach avoids the instability of episomal plasmids and the use of selection markers such as antibiotics. The CRISPR-Cas9 system, guided by a designed gRNA, cuts the target site of the gene cluster plasmid to be edited. Subsequently, repair is completed based on homologous recombination mediated by the λRed system (homologous arms are provided by the editing vector), achieving editing of the target site of the gene cluster.

[0046] This experiment also continued to delete recA based on BL22. At the same time, sg was knocked into the recA gene. T The module can express specific gRNA under IPTG induction to guide Cas9 to target and cut the editing plasmid, thereby removing the editing plasmid in the cell after editing is completed for subsequent iterative editing. T The strain of the module is named BL23. The construction process is described in Figure 3 .

[0047] The relevant plasmid and primer information used in this example and the following examples are shown in Tables 1 and 2.

[0048] Table 1: Plasmids used in the present invention

[0049]

[0050]

[0051]

[0052] Table 2: Primer sequences used in the present invention

[0053]

[0054]

[0055]

[0056] 1. Selection of Recipient Strain

[0057] Escherichia coli and yeast are two commonly used cell types for in vivo editing of gene cluster plasmids. Compared to E. coli, yeast has disadvantages such as slower growth, complex manipulation steps, the need for additional selection markers for exogenous DNA editing, and weak iterative editing capabilities. E. coli grows faster, is easier to manipulate, and has a mature CRISPR-Cas9-based gene editing system.

[0058] This example first tested the transformation efficiency of various E. coli strains, including TOP10, EPI300, DMS42, and BL21(DE3), for the large plasmid pCL01-omil: BAC plasmid pCL01 carrying 5-ketomilbemycin BGC (transformation efficiency is expressed as the average number of colonies produced per microgram of plasmid DNA transformed). The results showed that the BL21(DE3) strain had the highest transformation efficiency for the large plasmid ( Figure 2 ), so BL21(DE3) was selected as the starting strain for this study for subsequent modification to develop a method for scarless editing of intracellular gene cluster plasmids in E. coli. This not only solves the problem of transformation difficulties caused by low concentration of plasmid after cutting in extracellular editing methods, but also circumvents a series of problems such as cumbersome yeast cell operation, slow growth, and the need to introduce selection markers.

[0059] 2. Construction of BL22 strain

[0060] 2.1 Construction of knock-in plasmid (or editing vector)

[0061] To construct pTarget-Cas9+λRed / HA, we first need to construct a pTarget-HA (PmeI) plasmid containing sequences on both sides of the E. coli SS9 site (homologous arms, HA, each about 500 bp long), amplify sgRNA from pTargetF-cat using primers ins-gRNA F / gRNA R, amplify HA up (1166 bp) fragment using ins-HA up F / R as primers BL21 (DE3) as template, and amplify HA down (790 bp) fragment using ins-HA down F / R as primers BL21 (DE3) as template. The obtained three fragments sgRNA, HA up, and HA down were ligated with the enzyme-linearized vector pTargetF-cat (SpeI / HindIII) using multi-fragment recombination ligase 2× Universal CloneMix ligation was performed to obtain the plasmid pTarget-HA(PmeI). Subsequently, an 8200-bp fragment containing the Cas9 gene and the λRed system was amplified using ins-cas-red F / R primers and the pCas plasmid as a template. This fragment was then recombined with the linearized plasmid pTarget-HA(PmeI) digested with PmeI to obtain the editing vector pTarget-Cas9+λRed / HA plasmid.

[0062] 2.2 Construction of BL22 strain

[0063] The plasmid pCas (Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol. 2015; 81(7): 2506-14. doi: 10.1128 / AEM.04023-14.) was transformed into BL21(DE3) cells and cultured overnight at 30°C on LB solid medium plates. The next day, a single colony was picked and inoculated into 4 mL of liquid LB (added with kanamycin resistance) and cultured overnight at 30°C. 50 μL was transferred to a 50 mL shake flask to prepare BL21(DE3) / pCas electroporation competent cells. (Note: L-arabinose was added to the culture 30 min before preparing the competent cells to induce λRed expression, with a final concentration of 10 mM).

[0064] Take 100ng of editing plasmid pTarget-Cas9+λRed / HA and electroporate it into BL21(DE3) / pCas. After incubation for 1h, spread it on kanamycin and spectinomycin double-resistance LB plate and culture it at 30℃ overnight. The grown colonies were verified with primers F1 / R1 (expected to amplify a 1497bp fragment), F2 / R2 (1390bp), and F1 / R2 (10486bp). The correct colonies were inoculated into LB tubes and cultured at 30℃ overnight. Kanamycin was added, and 1mM IPTG was added to induce sgRNA expression to guide Cas9 to cut the editing plasmid, thereby removing the pTarget-Cas9+λRed / HA plasmid. The next day, the bacterial solution was diluted 10 -6Plate the plate onto a kanamycin-resistant plate and incubate overnight at 30°C. Once colonies have grown, inoculate them into liquid LB medium containing both spectinomycin and kanamycin and into liquid LB medium containing kanamycin, respectively, and incubate overnight at 30°C. If bacteria in the spectinomycin- and kanamycin-resistant tubes fail to grow, while bacteria in the corresponding kanamycin-resistant tubes grow normally, this indicates that the spectinomycin-resistant pTarget-Cas9+λRed / HA plasmid has been removed from the corresponding tubes.

[0065] Since pCas is a temperature-sensitive replication plasmid, it can replicate normally at 30°C, but cannot replicate at 37°C. Take 4 μL of the bacterial solution and transfer it to a 4 mL antibiotic-free LB tube and culture it at 37°C overnight. Then continue to take the bacterial solution and dilute it 10 -6 Spread the plate onto an antibiotic-free LB plate and incubate overnight at 37°C. Once colonies have grown, inoculate the corresponding plates into liquid LB medium containing kanamycin resistance and one containing antibiotics, and incubate overnight at 37°C. If the bacteria in the tube containing kanamycin fail to grow, it indicates that the kanamycin-resistant pCas plasmid has been removed. The bacteria in the antibiotic-free liquid LB medium are the strain that has successfully introduced the Cas9 and λRed system and removed the plasmid, and are designated BL22.

[0066] 3. Construction of strain BL23

[0067] 3.1 Editing plasmid pTarget-ΔrecA::sg T / HA construction process:

[0068] Construction of editing plasmid pTarget-ΔrecA::sg T / HA, using primers ΔrecA::sg T gRNA F / gRNA R, using pCas as a template, amplified the targeting sgRNA transcription cassette ΔrecA::sg T gRNA fragment; using ΔrecA::sg T up (upstream homology region) F / R primers were used to amplify the upstream homology arm ΔrecA::sg T up fragment; with ΔrecA::sg T down (downstream homology region) F / R were used as primers, and BL21 (DE3) was used as template to amplify the downstream homology arm recA::sg T down. T -F1 / R1 were used as primers and pCas was used as template to perform PCR to obtain sg T 1 fragment, in sg T -F2 / R2 as primers and pCas as template for PCR to obtain sg T 2 segments, using sgT -F1 / sg T -R2 is primer sg T 1 fragment and sg T sg was obtained by overlap PCR using 2 fragments as templates. T fragment, in which N20 targets the spectinomycin resistance gene. Using the four fragments obtained above as templates, the ΔrecA::sg T gRNA, ΔrecA::sg T up,sg T , recA::sg T down fragment sequence, using primers ΔrecA::sg T gRNA F / ΔrecA::sg T down R, and obtained ΔrecA::sg by overlap PCR T / HA fragment, and then ligated with the enzyme-digested plasmid pTargetF-cat (SpeI / HindIII) to obtain the editing vector pTarget-ΔrecA::sg T / HA.

[0069] 3.2 Construction of strain BL23

[0070] Plasmid pTarget-ΔrecA::sg T / HA was introduced into BL22 competent cells, and the recA gene was knocked out and sg T Module. By inducing its expression through IPTG, Cas9 can mediate the cutting of the spectinomycin resistance gene in the editing plasmid, thereby promoting the elimination of the editing plasmid. After the colonies grow, pick 20 colonies and use primers ΔrecA::sg T JF / R verification showed a 100% positive rate. Subsequent sequencing results further confirmed that sg T The module replaced the recA gene. In this study, the BL22 strain was used to T The strain with modular replacement of recA gene was named BL23.

[0071] Then, to verify the sg T To determine whether the module has the ability to eliminate the edited plasmid, the positive colonies were inoculated into a 4 mL LB tube, IPTG was added and cultured at 37°C overnight. -7Evenly spread onto an Abra-resistant LB plate, culture overnight at 37°C, and randomly pick 10 single colonies to be inoculated into a spectinomycin-resistant liquid LB tube and a non-resistance liquid LB tube. After 8 hours of culture, if the bacteria in the tube with spectinomycin do not grow (the liquid LB is clear), while the bacteria in the non-resistance tube grow (the liquid LB becomes turbid), it indicates that the pTarget-ΔrecA::sg T / HA plasmid has been removed. The experimental results show that all 10 colonies have successfully lost the plasmid, indicating that sg T The system has been knocked into the genome and can function well.

[0072] Example 2

[0073] Testing the editing ability of the strain BL23 obtained in Example 1 for 5-ketomilbemycin BGC

[0074] The plasmid pCL01-omil (Li L, Wei KK, Liu XC, Wu YJ, Zheng GS, Chen SX, Jiang WH, Lu YH (2019). aMSGE: advanced multiplex site-specific genome engineering with orthogonal modular recombinases in actinomycetes. Metab Eng, 52: 153-167) carrying 5-ketomilbemycin BGC was edited by three editing methods: knocking out the milE gene, inserting the milF gene, and replacing the milR gene promoter with the constitutive strong promoter kasOp*.

[0075] 1. Construction of editing vector pTarget-ΔmilE / HA

[0076] First, the milE gRNA fragment was amplified using primers milE gRNA F / gRNA R and pCas as a template. The milE up fragment was amplified using primers milE up F / R and pCL01-omil as a template. The milE down fragment was also amplified using primers milE down F / R and pCL01-omil as a template. Next, the ΔmilE-HA fragment was generated by overlap PCR using primers milE gRNA F / milE down R and the milE gRNA, milE up, and milE down fragments as templates. This fragment was then recombined and ligated with the linearized vector pTargetF-cat (SpeI / HindIII) to generate the plasmid pTarget-ΔmilE / HA.

[0077] 2. Construction of editing vector pTarget-::milF / HA

[0078] A similar method was used to obtain the milF gRNA fragment; using milF F / R as primers and the Streptomyces hygroscopicus SIPI-054 genome as a template, the fragment to be knocked in, "::milF-HA," was obtained by overlap PCR using milF gRNAF / milF R as primers and the milF gRNA fragment and ::milF-HA as templates. This fragment was then ligated with the linearized plasmid pTargetF-cat (SpeI / HindIII) to generate the plasmid pTarget-::milF / HA.

[0079] 3. Construction of editing vector pTarget-milR-kasOp* / HA

[0080] Similarly, the milR-kasOp*gRNA fragment, milR-kasOp*up fragment, and milR-kasOp*down fragment were obtained respectively. The kasOp* promoter was obtained by PCR amplification using primers kasOp*F / R and a plasmid containing the kasOp* promoter fragment as a template. Then, using primers milR-kasOp*gRNA F / milR-kasOp*down R, and the milR-kasOp*gRNA fragment, milR-kasOp*up fragment, kaso*p promoter fragment, and milR-kasOp*down fragment as templates, milR-kasOp* / HA was obtained by overlap PCR. It was then ligated with the enzyme-linearized plasmid pTargetF-cat (SpeI / HindIII) to obtain the plasmid pTarget-milR-kasOp* / HA.

[0081] 4. Editing the 5-ketomilbemycin BGC plasmid pCL01-omil

[0082] First, the pCL01-omil plasmid was introduced into BL23, and the successfully transformed BL23 / pCL01-omil strain was prepared into a competent state. Then 100 ng of the constructed editing vector pTarget-ΔmilE / HA was electroporated and introduced into the BL23 / pCL01-omil competent state. The transformation system was evenly spread on a solid LB plate and cultured at 37°C overnight. After the colonies grew, PCR verification was performed using the corresponding primers milE JF / R, milF JF / R, and milR-kasOp*JF / R. Then, the same method was used to complete the knock-in of the milF gene using the plasmid pTarget-::milF / HA, and the replacement of the milR promoter with kasOp* using the pTarget-milR-kasOp* / HA plasmid. The results showed that the efficiencies of the knockout of the milE gene, the knock-in of the milF gene, and the replacement of the milR promoter using the iCASRED method developed in this study were 93.3%, 27.4%, and 79.8%, respectively (see Figure 4 ), and the positive PCR products were verified to be correct by sequencing (sequencing results see Figure 5 ).

[0083] Example 3

[0084] Functional verification of the gene cluster after editing by the method of Example 2 (verified by heterologous expression in Streptomyces avermitilis chassis cells and multi-copy expression in Streptomyces milbeyi)

[0085] 1. Construction of avermectin BGC deletion mutant.

[0086] First, the avermectin BGC knockout plasmid pKCCas9ΔAve-HA (pSG5, temperature-sensitive replicon) was constructed, and then the avermectin biosynthetic gene cluster was knocked out according to the one-step high-efficiency CRISPR / Cas9 method (Huang H, Zheng G, Jiang W, HuH, Lu Y. One-step high-efficiency CRISPR / Cas9-mediated genome editing in Streptomyces. Acta Biochim Biophys Sin (Shanghai). 2015, 47(4): 231-43. doi: 10.1093 / abbs / gmv007.). The avermectin gene cluster deletion mutant strain MSB01Δave of Streptomyces avermectin MSB01 was constructed.

[0087] 2. Functional Verification of the pCL01-omil::milF Editing Plasmid

[0088] First, the pCL01-omil::milF plasmid containing the milF gene for the milbemycin BGC was introduced into the avermectin-deficient chassis strain MSB01Δave and the milbemycin strain SIPI-054 by conjugation, generating the MSB01Δave::mil and SIPI-054::mil strains, respectively. Both strains were streaked onto MS solid medium and cultured in a 30°C incubator for 6 days. Then, an inoculation shovel was used to dig out approximately 1 cm of 2 10% of the seeds were inoculated into 30 mL of fermentation medium (50 g / L corn starch, 10 g / L soybean meal, 10 g / L cottonseed meal, 0.1 g / L α-amylase, 5 g / L sodium chloride, 7 g / L calcium carbonate, pH = 7.1 ± 0.1) and fermented at 28°C and 220 rpm for 10 days.

[0089] 3. HPLC Analysis of Milbemycin

[0090] After fermentation, 500 μL of the fermentation sample was placed in an EP tube, 1.5 mL of anhydrous ethanol was added, ultrasonic extraction was performed for 30 minutes, centrifuged at 12000 rpm for 5 minutes, and an appropriate amount of the supernatant was taken and filtered through a 0.45 μm microporous organic filter membrane. Finally, the content of milbemycin A3 / A4 or 5-ketomilbemycin A3 / A4 was quantified by HPLC (see Figure 6HPLC (Shimadzu LC-20AT) conditions were as follows: a C18 column (4.6 mm × 150 mm, 5 μm), a mobile phase consisting of acetonitrile / water (volume ratio 73:27), a flow rate of 1.2 mL / min, a column temperature of 30°C, UV detection at 240 nm, and an injection volume of 10 μL. The retention times of milbemycins A3 and A4 were 10.2 and 12.7 min, respectively, and those of 5-ketomilbemycins A3 and A4 were 11.8 and 15.3 min, respectively.

Claims

1. A chassis strain for traceless editing of gene clusters in vivo, characterized in that: It is obtained by simultaneously integrating the Cas9 gene and the λRed recombination system into the genome of the recipient strain.

2. A chassis strain for seamless editing of gene clusters in vivo according to claim 1, characterized in that: The inducible sgRNA expression cassette (sg T ).

3. A chassis strain for seamless editing of gene clusters in vivo according to claim 1, characterized in that: The Cas9 gene is Cas9 from Streptococcus pyogenes.

4. A chassis strain for seamless editing of gene clusters in vivo according to claim 1, characterized in that: The recipient strain is Escherichia coli.

5. A chassis strain for seamless editing of gene clusters in vivo according to claim 4, characterized in that: The recipient strain is the Escherichia coli BL21 (DE3) strain.

6. A chassis strain for seamless editing of gene clusters in vivo according to claim 4, characterized in that: The recA gene was knocked out from the genome of Escherichia coli.

7. A method for constructing a chassis strain for traceless editing of gene clusters in vivo, characterized in that: The following steps are included: S-1. The sequences on both sides of the E. coli SS9 site, the Cas9 gene and the λRed recombination system were constructed into the vector pTargetF-cat to obtain the editing vector pTarget-Cas9+λRed / HA plasmid; S-2. Using the dual plasmid editing system of E. coli, the Cas9 gene and λRed recombination system were knocked into the genome of E. coli; S-3. Based on the transformed E. coli obtained in step S-2, the recA gene in the genome of E. coli is further knocked out, and sg is knocked in at the knockout site. T The system thus obtained a chassis strain for scarless editing of gene clusters in vivo.

8. The construction method according to claim 7, characterized in that: The operation of step S-1 is as follows: first, construct a pTarget-HA plasmid containing homologous arms on both sides of the Escherichia coli SS9 site, amplify sgRNA from pTargetF-cat using primers ins-gRNA F / gRNAR, amplify HA up fragment using ins-HA up F / R as primers and BL21 (DE3) as template, amplify HA down fragment using ins-HA down F / R as primers and BL21 (DE3) as template, connect the three obtained fragments sgRNA, HA up, and HA down with the enzyme-cut linearized vector pTargetF-cat to obtain plasmid pTarget-HA, and then recombinantly connect the fragments of Cas9 gene and λRed recombination system with plasmid pTarget-HA to obtain pTarget-Cas9+λRed / HA plasmid.

9. The construction method according to claim 7, characterized in that: The operation of step S-2 is as follows: electroporate the pCas of the E. coli dual plasmid editing system carrying the Cas9 gene and the λRed system into BL21(DE3), and prepare the BL21(DE3) / pCas competent cells; The editing vector pTarget-Cas9+λRed / HA plasmid was electroporated into BL21(DE3) / pCas competent cells, plated on kanamycin and spectinomycin double-resistant LB plates, and cultured overnight; the grown colonies were verified, and the verified correct colonies were inoculated into LB test tubes for overnight culture, kanamycin was added, and IPTG was added to induce the expression of sgRNA to cut the editing plasmid, thereby removing the editing vector pTarget-Cas9+λRed / HA; the plasmid pCas (containing a thermosensitive promoter) was removed by passage at 37°C to obtain the modified Escherichia coli (BL23) with the knock-in Cas9 gene and λRed system.

10. A method for seamless editing of a gene cluster in vivo, characterized in that: Use the chassis strain described in any one of claims 1-6 or the chassis strain obtained by the construction method described in any one of claims 7-9.

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