CN115786229A - Recombination system for knockout of rhodococcus gene and application thereof - Google Patents
Recombination system for knockout of rhodococcus gene and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of cell engineering, and particularly relates to a recombinant system for knockout of rhodococcus genes and application thereof. The method comprises the following steps: the Che9c60&61 recombinase plasmid, flp recombinase and free double-stranded DNA knockout cassette fragments; the free double-stranded DNA knockout cassette fragment comprises: upstream and downstream homology arm sequences of a target gene and two homodromous FRT sequences containing resistance genes are inserted between the upstream and downstream homology arm sequences; the gene sequence of the Flp recombinase plasmid is shown as SEQ ID NO.1 or SEQ ID NO. 3. The recombination system for knocking out the rhodococcus gene can effectively knock out the rhodococcus tracelessly, and the loss efficiency of the gentamicin resistance reaches 100 percent and 78.7 percent; moreover, the invention can effectively realize the efficient traceless knockout of Rhodococcus ruber R1 based on the FRT site-specific recombinase Flpw method, and recover the resistance gene for the next round of gene editing (multigene knockout).
Description
Technical Field
The invention belongs to the technical field of cell engineering, and particularly relates to a recombinant system for knockout of rhodococcus gene and application thereof.
Background
Rhodococcus (Rhodococcus) is an aerobic, nonmotile, spore-free and mycolic acid-containing gram-positive bacterium, which is intermediate between Mycobacterium (Mycobacterium), nocardia (Nocardia) and Corynebacterium (Corynebacterium). Rhodococcus is widely distributed in different environments from sea to land, can adapt to severe environment, and has catabolic activity of various organic compounds, such as monoaromatic hydrocarbon, polyaromatic hydrocarbon, phenol, halogenated hydrocarbon, nitroaromatic hydrocarbon, nitrile and other aromatic compounds. Although the potential application of Rhodococcus in the fields of biodegradation, transformation, synthesis and the like is recognized, the quantity of the basic research of Rhodococcus is far less than that of the more mature engineering bacteria such as Escherichia coli and Bacillus. Rhodococcus was not well understood. The gene knockout tool of the rhodococcus is very important for people to carry out basic research on the rhodococcus.
At present, few gene knockout tools are available for rhodococcus, and the existing traditional homologous recombination technology has the characteristic of low recombination efficiency in gene knockout of rhodococcus. The existing research finds that the rhodococcus gene can be accurately and efficiently edited by a mycobacterium phage recombinase Che9c60&61, but the antibiotic resistance gene can be introduced in the gene knockout process in such a way, so that multiple rounds of gene editing cannot be carried out.
Therefore, it is necessary to develop a recombination system which has high recombination efficiency and can be used for knockout of Rhodococcus genes by performing multiple rounds of gene editing.
Disclosure of Invention
In order to solve the above problems, it is an object of the present invention to provide a recombination system for knockout of a rhodococcus gene. The recombination system can effectively perform efficient traceless knockout on rhodococcus based on an FRT site-specific recombinase Flpw method, and recovers a resistance gene for the next round of gene editing (multigene knockout).
In order to achieve the purpose, the invention can adopt the following technical scheme:
in one aspect, the present invention provides a recombination system for knockout of a rhodococcus gene, comprising: the Che9c60&61 recombinase plasmid, flp recombinase and free double-stranded DNA knockout cassette fragments; the free double-stranded DNA knockout cassette fragment comprises: the upstream and downstream homology arm sequences of a target gene are inserted with two homodromous FRT sequences containing resistance genes; the gene sequence of the Flp recombinase plasmid is shown as SEQ ID NO.1 or SEQ ID NO. 3.
The invention also provides a construction method of the genetic engineering rhodococcus, which comprises the following steps: the recombinant system for knocking out the rhodococcus gene is used for knocking out the rhodococcus gene to obtain the genetically engineered rhodococcus.
The invention further provides the genetically engineered rhodococcus constructed by the construction method of the genetically engineered rhodococcus.
In another aspect, the invention provides a Flp recombinase gene sequence, which comprises a sequence shown as SEQ ID NO.1 or SEQ ID NO. 3.
In another aspect, the invention provides a recombinant system for knocking out rhodococcus genes or an application of the Flp recombinase gene sequence in rhodococcus gene editing.
The beneficial effects of the invention include: the recombination system for knocking out the rhodococcus gene can effectively knock out the rhodococcus tracelessly, and the loss efficiency of the gentamicin resistance reaches 100 percent and 78.7 percent; moreover, the invention can effectively realize the efficient traceless knockout of Rhodococcus ruber R1 based on the FRT site-specific recombinase Flpw method, and recover the resistance gene for the next round of gene editing (multigene knockout).
Drawings
FIG. 1 is a schematic illustration of the construction of a fragment of the amiE2 knock-out cassette;
FIG. 2 is a schematic diagram of pBNVW plasmid construction;
FIG. 3 shows PCR verification of Rhodococcus ruber R1 and its mutant;
FIG. 4 shows sequencing verification of Rhodococcus ruber R1 and its mutant;
FIG. 5 shows the screening of resistant missing spots of Rhodococcus ruber 1. DELTA. AmiE2 FRT-Gm-FRT;
in fig. 3, note: lanes 1, 2 and 3 are DNA fragments amplified using Rhodococcus ruber R1 and Rhodococcus ruber R1. DELTA. AmiE2 as templates and using the primers amiE2-P9/P10 as templates, respectively; m represents Marker.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context has a significantly different meaning, the singular forms of expressions include the plural forms of expressions. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to refer to the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may be present or may be added. As used herein, "/" can be interpreted as "and" or "depending on the situation.
An embodiment of the present invention provides a recombination system for knockout of a rhodococcus gene, comprising: the Che9c60&61 recombinase plasmid, flp recombinase and free double-stranded DNA knockout cassette fragments; the free double-stranded DNA knockout cassette fragment comprises: the upstream and downstream homology arm sequences of a target gene are inserted with two homodromous FRT sequences containing resistance genes; the gene sequence of the Flp recombinase plasmid is shown as SEQ ID NO.1 or SEQ ID NO. 3.
It is to be noted that the sequence shown in SEQ ID NO.1 is a flp gene sequence, the sequence shown in SEQ ID NO.3 is subjected to rhodococcus codon optimization (also called flpw gene), and a constitutive PamiC promoter sequence is added before the start codon of the flpw gene; an Xba I cleavage site was designed at the 5 'end of Pamic and a Kpn I cleavage site was designed at the 3' end of flpw to give a 5'Xba I-Pamic-flpw-Kpn I3' sequence (SEQ ID NO. 3).
It should be further noted that the invention finds that the purpose of traceless knockout and multi-round gene editing can be achieved by introducing the same-direction FRT sites at the two ends of the antibiotic resistance gene and then accurately and efficiently recombining the two same-direction FRT sites by using the site-specific recombinase Flp, so that the resistance gene between the FRT sites is lost.
In the embodiment of the present invention, pUC57 is used as a vector to construct a plasmid carrying the knockout cassette fragment, and the plasmid is amplified by PCR or digested by restriction enzymes to obtain the desired free double-stranded DNA knockout cassette fragment. This fragment was used in a recombination system for traceless gene knockout, and the feasibility of the system was verified in Rhodococcus ruber R1.
In some embodiments, in the above recombinant system for rhodococcus gene knock-out, the resistance gene comprises a gentamicin resistance gene. It is noted that the resistance gene may be known in the art, such as the gentamicin resistance gene.
Another embodiment of the present invention provides a method for constructing a genetically engineered rhodococcus, comprising: and (3) knocking out genes of the rhodococcus by using the recombination system for knocking out genes of the rhodococcus to obtain the genetically engineered rhodococcus. The Rhodococcus is preferably Rhodococcus ruber R1; the rhodococcus gene is preferably Δ amiE2.
In some embodiments, the method for constructing the genetically engineered rhodococcus includes: over-expressing the Che9c60&61 recombinase gene in a host, and recombining a sequence containing a resistance gene between two directional FRTs to a target knockout gene of rhodococcus by using a homologous recombination method; and then, loss of the resistance gene is realized by using optimized Flp recombinase.
The invention further provides the genetically engineered rhodococcus constructed by the construction method of the genetically engineered rhodococcus.
In another embodiment, the invention provides a Flp recombinase gene sequence, which comprises a sequence shown as SEQ ID NO.1 or SEQ ID NO. 3. It should be noted that the recombinase encoded by the Flp recombinase gene sequence of the invention can mediate efficient recombination of two directional FRT sites at the knockout position of a target gene in the rhodococcus genome.
In another embodiment of the present invention, a recombinant system for knocking out a rhodococcus gene or an application of the Flp recombinase gene sequence in rhodococcus gene editing is provided. The recombination system for knocking out a rhodococcus gene or the Flp recombinase gene sequence can be used for knocking out a rhodococcus gene, so that the recombination efficiency is high, and multiple rounds of gene editing can be performed.
For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples.
In the embodiment of the present invention, rhodococcus ruber R1 and Escherichia coli Dh5 alpha are used as the unit for preservation; the plasmids pRCTc-pa2-che9c60&61, pBNVCm, pUC57, pKD3, pSRKGm and pSRKKm used were deposited as units; restriction enzymes, ligases and other reagents were purchased from Baori doctor technology (Beijing) Ltd; one-step rapid cloning and Taq enzyme was purchased from Kyoto Jinsha Biotech, inc.
In the embodiment of the invention, the PCR primer is synthesized by Guangzhou Aiji Biotechnology Co., ltd; plasmids pBNVW and pUC57W1 were constructed for the present invention.
In the embodiment of the invention, amidase gene amiE2 is selected as a knockout target gene, and the acrylamide yield of the strain can be improved after the gene is knocked out.
In the examples of the present invention, the primer pairs used are shown in Table 1 below (the underlined portions are enzyme cleavage sites).
TABLE 1 primer sequences
In the embodiment of the invention, rhodococcus ruber R1 delta amiE2 comprises FRT-Gm-FRT (SEQ ID NO. 4) and a Rhodococcus ruber 1 delta amiE2 sequence (SEQ ID NO. 5) recombined by FRT, wherein the underline is an amiE2-P9/P10 primer sequence, the capital letters are amiE2 upstream and downstream gene sequences, the lowercase letters are knockout box non-homologous arm sequences, and a character shading and framing region is an FRT sequence:
SEQ ID NO.4(Rhodococcus ruber R1ΔamiE2::FRT-Gm-FRT)：
SEQ ID NO.5(Rhodococcus ruber R1 ΔamiE2)：
(1) Rhodococcus ruber 1/pRCTc-pa2-che9c60&61 construction
To make pRCTc-pa2-che9c60&61 (Liang et al. Metabolic Engineering,2020, 57. Rhodococcus ruber 1 was mixed with 10mL of a shock-ready solution (20 g of glucose, 5g of yeast powder, 0.655g of K) 2 HPO 4 ·3H 2 O、0.5g KH 2 PO 4 、0.5g MgSO 4 ·7H 2 O, 8.5g of glycine and 0.0015g of isoniazid, and the volume is determined to be 1L) by double distilled water;
in addition, when double-stranded DNA knockout cassette fragments are converted by electric shock, 5mg of urea needs to be added into the 10mL of electric shock preparation solution, the mixture is cultured for 2d-3d at 30 ℃ and 200rpm, 1mL of bacterial solution is taken to ice for precooling for 5min, the mixture is centrifuged at 6000rpm for 5min at 4 ℃, supernatant is removed, and the precipitate is resuspended by 1mL of 10% (V/V) glycerol precooled at 4 ℃, and the step is repeated for 3 times; after the supernatant is removed by the last centrifugation, the precipitate is resuspended by 100 mu L of 10% glycerol, about 1 mu g of free double-stranded DNA knockout cassette fragment is added, and the mixture is kept stand on ice for 5min; adding 100 mu L of bacterial liquid of the mixed plasmid into a 1mm electric shock cup, and setting the voltage 1250V of an electric converter for electric shock conversion; 100 mu L of the bacterial liquid in the electric shock cup is taken out to a centrifugal tube of 1.5mL, 900 mu L of electric shock resuscitation liquid (5 peptone, 5g sodium chloride, 2.5g yeast extract, 18.5g brain heart infusion and 91g sorbitol are added, double distilled water is added to the mixture until the volume is 1L), the mixture is cultured for 3h-4h at the temperature of 30 ℃ and 200rpm, and the mixture is coated on an LB plate of tetracycline (10 mu g/mL) to be cultured for 3d-4d at the temperature of 30 ℃.
The colony PCR is verified to obtain Rhodococcus ruber R1/pRCTc-pa2-che9c60&61, and competent cells are prepared by the method for preparing shock competence and are used for shock transformation of double-stranded DNA knockout cassette fragments.
(2) Construction of pUC57-amiE2up-down plasmid
Firstly, constructing a plasmid containing an amiE2 gene knockout cassette by using pUC57 as a vector: pUC57W1 (the knockout cassette construction process is shown in FIG. 1). Primers were designed using the amiE2 gene of strain Rhodococcus ruber 1: amiE2-P1/P2/P3/P4/P5/P6/P7/P8 (see Table 1); the up-and-down-stream approximately 700bp fragments of the amiE2 gene were obtained by amplification with the amiE2-P5/P2 and amiE2-P6/P3 primer fragments, respectively (50. Mu.L amplification systems: 2. Mu.L each of the amiE2-P5/P2 or amiE2-P6/P3 primer fragments, 1. Mu.L of Rhodococcus ruber R1 total DNA, 45. Mu.L of S4 Fidelity PCR Mix; amplification conditions: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 15S; program cycle number: 35 cycles).
In the embodiment of the invention, when primers are designed, a short overlapping extension connector comprising SpeI and BamHI enzyme cutting sites is designed at the tail end of an amiE2-P2/P3 primer in advance, two fragments of 700bp and 900bp obtained by amplification are used as primers for an overlapping extension PCR method, and the overlapping extension PCR is used for amplifying for 10 cycles (a 25 mu L amplification system is that 2 mu L of each of the two fragments of 700bp and 19 mu L of S4 Fidelity PCR Mix; the amplification conditions are that the temperature is 98 ℃ for pre-denaturation 2min, the temperature is 98 ℃ for denaturation 10s, the temperature is 54 ℃ for annealing 15s, the temperature is 72 ℃ for extension 15S, and the program cycle number is 10 cycles);
then, the overlapping extension PCR product is used as a template, and the amiE2-P1/P4 is used as a primer for amplification, so that the upstream gene and the downstream gene of the amiE2 gene can be spliced together by about 500bp respectively (a 50 mu L amplification system: 2 mu L of each amiE2-P1/P4 primer fragment, 1 mu L of overlapping extension PCR product, and 45 mu L of S4 Fidelity PCR Mix; amplification conditions: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 15s, and extension at 72 ℃ for 25S; program cycle number: 35 cycles);
after the fragments after the overlap extension were digested with restriction enzymes HindIII and EcoRI (the digestion conditions were: 50. Mu.L of the fragments and plasmid, 10 Xbuffer 10. Mu.L, hindIII 2. Mu.L, and EcoRI 2. Mu.L, double distilled water was supplied to 100. Mu.L, and the mixture was reacted at 37 ℃ for 2 hours), ligated to pUC57 digested with the same restriction enzymes (the ligation system was: 10 Xligase buffer 1. Mu.L, after digestion, 6. Mu.L of the fragments, 2. Mu.L of the vector after digestion, and 1. Mu.L of T4 DNA ligase to 20. Mu.L, and ligated at 16 ℃ for 12 hours), heat-shock-transformed Dh 5. Alpha. And confirmed by colony PCR, a pUCupUCp 57-amiE 2-down plasmid was obtained.
(3) Construction of a plasmid carrying a sequence comprising a gentamicin resistance Gene between two homeotropic FRTs
PCR amplification is carried out by taking an FRT sequence in pKD3 as a template and FRT-P1/P2/P3/P4 as a primer to obtain two fragments (FRTup and FRTdown), and pSRKKm-FRTup-down plasmid is constructed by a one-step rapid Cloning method (one-step rapid Cloning system is pSRKKm 1 muL, FRTup and FRTdown are respectively 2 muL, 2 x Uniclone SeamLess Cloning Mix is 5 muL; the reaction condition is that the mixture is treated at 50 ℃ for 20min and is iced for 2 min); after heat shock transformation of Dh5 alpha and colony PCR verification, pSRKKm-FRTup-down plasmid was obtained.
The gentamicin resistant gene sequence in the pSRKGm plasmid is used as a template, gm-P1/P2 is used as a primer for PCR amplification to obtain a gentamicin resistant gene fragment, the obtained gentamicin resistant gene fragment is recombined on the pSRKKm-FRTup-down plasmid by using a one-step rapid Cloning method (the one-step rapid Cloning system is pSRKKm-FRTup-down 1.5 muL, the gentamicin resistant gene fragment is 3.5 muL, and 2 x Uniclone SeamLess Cloning Mix is 5 muL, the reaction condition is that the plasmid is treated at 50 ℃ for 20min and is iced for 2 min), and the plasmid pSRKKm-FRT-Gm-FRT is obtained after Dh5 alpha is thermally shock transformed and colony PCR verification.
(4) Rhodococcus ruber 1. DELTA. AmiE2: FRT-Gm-FRT/pRCTc-pa2-che9c60&61 mutant construction
pSRKKm-FRT-Gm-FRT and pUC57-amiE2up-down are used for designing a one-step rapid cloning primer amiE2-FRT-Gm-P1/P2, a 1142bp fragment is obtained by PCR amplification, and a pUC57W1 plasmid with the FRT-Gm-FRT fragment inserted in the middle of pUC57-amiE2up-down is obtained by one-step rapid cloning according to the method. Free double-stranded DNA knock-out cassette fragments of the amiE2 gene were obtained by PCR amplification of the amiE2-P7/P8 primers or digestion of pUC57W1 with the restriction enzymes Hind III and EcoRI.
After transformation of the amiE2 double-stranded DNA knock-out cassette fragment to Rhodococcus ruber R1/pRCTc-pa2-che9c60&61 by electric shock, colony PCR and sequencing validation using the amiE2-P9/P10 primers (results shown in FIGS. 3 and 4) gave Rhodococcus ruber 1. Delta. AmiE2:: FRT-Gm-FRT/pRCTc-pa2-che9c60&61 mutant (see SEQ ID NO. 4).
(5) pBNVW plasmid construction
In order to obtain a plasmid which has a high-efficiency plasmid exit mechanism and carries a Flpw recombinase, pBNVCm plasmid carrying a temperature-sensitive replicon pB264 which can exit at 37 ℃ with high efficiency is taken as a vector, and the recombinase Flpw (the name of the recombinant plasmid is pBNVW) is connected and optimized. The Nanjing Kingsry Biotechnology Ltd was entrusted with Rhodococcus codon optimization of the amino acid sequence (SEQ ID NO. 2) of recombinase Flp encoded by Flp gene (SEQ ID NO. 1) (the gene obtained by optimization was named flpw gene), and a constitutive PamiC promoter sequence was added before the start codon of flpw gene. An Xba I cleavage site was designed at the 5 'end of Pamic, a KpnI cleavage site was designed at the 3' end of flpw, and a 5'Xba I-Pamic-flpw-KpnI 3' sequence (see SEQ ID NO. 3) was synthesized by Nanjing King Korea Biotech Co., ltd and recombined into pBNVCm vector through the same cleavage sites to obtain pBNVW plasmid (the plasmid construction scheme is shown in FIG. 2).
For traceless knock-out purposes, recombinant Rhodococcus ruber R1. DELTA. AmiE2, two homeotropic FRT sites of FRT-Gm-FRT, were recombined using Flpw recombinase to lose gentamicin resistance, for which purpose it was necessary to prepare shockable competent cells of Rhodococcus ruber 1. DELTA. AmiE2, FRT-Gm-FRT.
Rho preparation using the above method for preparing shock competencedococcus ruber R1ΔamiE2::FRT-Gm-FRT/pRCTc-pa2-che9c60&61 shock-competent cells. After the completion of the electroporation transformation, colony PCR verification was performed using pB264-P1/P2 primers. The verified Rhodococcus ruber R1. Delta. AmiE 2:FRT-Gm-FRT/pRCTc-pa 2-che9c60&61/pBNVW is cultured in 5mL liquid LB medium at 30 deg.C and 200rpm for 2-3d, 100 μ L bacterial liquid is taken and diluted to 10% -6 Spread on an LB plate with tetracycline and chloramphenicol (15. Mu.g/mL) for 3-4d. Single colonies are picked by using an aseptic toothpick and sequentially spotted on a gentamicin LB plate and a chloramphenicol LB plate, and because the efficiency of gentamicin resistance loss is different due to the action of a Flpw recombinase on an FRT locus, two strains with no recombination on the FRT locus and the recombination on the FRT locus can be generated. Strains that could not grow on gentamicin LB plates were Rhodococcus ruber R1. DELTA. AmiE2 mutants (see SEQ ID No. 5) that recombined at the FRT site, losing gentamicin resistance.
(6) Flpw efficiency validation
Colony PCR and sequencing validation were performed using the amiE2-P9/P10 primers (results are shown in FIGS. 3 and 4) to obtain Rhodococcus ruber 1. Delta. AmiE2/pRCTc-pa2-che9c60&61/pBNVW. In order to verify the efficiency of Flpw, rhodococcus ruber R1. Delta. AmiE2 strain was subjected to two shock transformations of pBNVW in the example of the invention, and 3 pBNVW positive transformants were picked for each shock transformation and subjected to resistance negative screening according to the above experimental procedures and data recording. Wherein the loss efficiency of gentamicin resistance is the expression of Flpw on the recombination efficiency of the FRT site in Rhodococcus ruber R1. DELTA. AmiE2. FRT-Gm-FRT, which represents the efficiency of the optimized recombinase Flpw in Rhodococcus ruber R1 (statistical data results are shown in FIG. 5 and Table 2). From FIG. 5 and Table 1, it can be seen that two shocks of pBNVW mediated Rhodococcus ruber R1. DELTA. AmiE2: traceless knockout of FRT-Gm-FRT strain, the loss efficiency of gentamicin resistance reached 100% and 78.7%, and sequence analysis of the mutant showed that Flpw mediated recombination between two FRT sites, thereby deleting the gentamicin resistance gene between the FRT sites, leaving only one FRT site (see SEQ ID NO. 5). This shows that the established FRT site-specific recombinase Flpw-based method can realize efficient traceless knockout of Rhodococcus ruber 1, and recover resistance genes for the next round of gene editing (multigene knockout).
TABLE 2 optimization of the efficiency of the recombinase Flpw in Rhodococcus ruber R1
Finally, the above embodiments are only intended to illustrate the technical solution of the present invention and not to limit the same, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, which shall be covered by the claims of the present invention.
Claims (10)
1. A recombination system for knockout of a rhodococcus gene, comprising: the Che9c60&61 recombinase plasmid, flp recombinase and free double-stranded DNA knockout cassette fragments; the free double-stranded DNA knockout cassette fragment comprises: the upstream and downstream homology arm sequences of a target gene are inserted with two homodromous FRT sequences containing resistance genes; the gene sequence of the Flp recombinase plasmid is shown in SEQ ID NO.1 or SEQ ID NO. 3.
2. The recombinant system for rhodococcus gene knockout of claim 1, wherein the resistance gene comprises a gentamicin resistance gene.
3. A construction method of genetic engineering Rhodococcus is characterized by comprising the following steps: genetically engineered Rhodococcus is obtained by gene-knocking out Rhodococcus using the recombinant system for Rhodococcus gene-knocking out according to claim 1 or 2.
4. The method according to claim 3, wherein the Rhodococcus is Rhodococcus ruber R1.
5. The method according to claim 4, wherein the Rhodococcus gene is Δ amiE2.
6. The building method according to claim 3, comprising: over-expressing the Che9c60&61 recombinase gene in a host, and recombining a sequence containing a resistance gene between two directional FRTs to a target knockout gene of rhodococcus by using a homologous recombination method; and then, loss of the resistance gene is realized by using optimized Flp recombinase.
7. The building method according to claim 4 or 5, comprising: over-expressing the Che9c60&61 recombinase gene in a host, and recombining a sequence containing a resistance gene between two directional FRTs to a target knockout gene of rhodococcus by using a homologous recombination method; and then the loss of the resistance gene is realized by using the optimized Flp recombinase.
8. The genetically engineered Rhodococcus constructed by the method of constructing genetically engineered Rhodococcus according to any one of claims 4 to 7.
9. A Flp recombinase gene sequence is characterized by comprising a sequence shown as SEQ ID NO.1 or SEQ ID NO. 3.
10. The recombinant system for knocking out rhodococcus gene according to claim 1 or 2 or the use of the Flp recombinase gene sequence according to claim 8 for rhodococcus gene editing.
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