CN111349641A - Genome integration method and application - Google Patents
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Abstract
The invention discloses a genome integration method and application. The Escherichia coli genome integration vector comprises a replicon, a target exogenous metabolic pathway, a resistance gene and an integration site, wherein the integration site IS an IS sequence; the engineering bacteria comprise Escherichia coli and the integration vector integrated into Escherichia coli genome. The invention uses IS sequence with more copy number in the Escherichia coli genome as integration site to integrate the vector and the genome, not only the integration method IS simple, but also the target gene integrated on the genome IS stable in heredity, thereby not only solving the problem of unstable separation when the plasmid IS used as an expression vector, but also simplifying the existing integration technology, and solving the problems of fussy and time-consuming existing large fragment integration technology.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and biology, relates to a genome integration method and application, and particularly relates to an escherichia coli large-fragment genome integration vector and application thereof in carotenoid synthesis.
Background
Because the Escherichia coli gene is simple and convenient to operate, a plurality of commercial alternative expression vectors are provided, and different promoters and replicons with different copy numbers are convenient for the expression of the protein in the Escherichia coli; therefore, for the metabolic engineering strategy of E.coli, the use of plasmids as expression vectors is currently in widespread use. The presence of plasmids, however, often leads to an excessive metabolic burden, instability of isolation and uncontrollable expression of the protein; meanwhile, in order to maintain the stable existence of plasmids in strains, antibiotics are generally required to be added, which not only increases the production cost, but also brings about the problem of drug resistance, and threatens human health. Therefore, genomic integration represents a great advantage.
Currently, genomic integration is performed in E.coli, mainly by homologous recombination, site-directed integration and transposase-mediated recombination. The most common homologous recombination is lambda-Red recombination, but in the technology, a proper recombination site needs to be searched when each integration is carried out, and then different homologous arms are designed according to the integration site, so that the time and the labor are consumed; moreover, as the length of the target gene increases, the recombination efficiency will be greatly reduced, and the length of the integrated gene is greatly limited. Site-directed integration is often performed by means of a phage integration site within E.coli. Transposase-mediated recombination needs to treat target genes by using transposase and then carry out random integration, and experimental operation is complicated. Some researchers use FRT sites as integration sites to perform multi-copy integration, but the method is simple, but the resistance genes cannot be deleted, so that the metabolic burden of cells is increased, and the biological safety problem is possibly brought. At present, various genome integration methods based on lambda-Red recombination are developed for the integration of large fragment genes, wherein the CRISPR-Cas9 technology is widely applied. However, the upper limit of the integration length is only 10kb, and the editing efficiency is rapidly reduced with the increase of the length of the integrated fragment. Therefore, it is important to develop a safe and simple method for integrating large segments of E.coli genome.
Disclosure of Invention
The invention aims to provide an escherichia coli large-fragment genome integration vector and application thereof in carotenoid synthesis, the integration vector can integrate large-fragment exogenous approaches into an escherichia coli genome, the integration method is simple and stable in heredity, and the integration efficiency can reach 100%.
An integration vector of an escherichia coli genome comprises a replicon, a target exogenous metabolic pathway, a resistance gene and an integration site, wherein the target exogenous metabolic pathway comprises a speed-limiting step in an MEP pathway and a lycopene synthesis gene.
The rate limiting step in the MEP pathway comprises a feed forward control module dxs-dxr and a feedback control module idi-crtE, wherein the dxs-dxr module is under a phoR promoter, and the idi-crtE module is under a yejG promoter.
The lycopene synthesis gene is under the trc promoter. Lycopene synthesis genes include the crtE, crtB and crtI genes.
The integration site IS an IS sequence; the replicon is R6K; the resistance gene is a chloramphenicol resistance gene.
Preferably, the IS sequence IS an IS5 sequence.
IS sequences, the simplest transposable element, are a normal component of many bacterial chromosomes, particularly E.coli, which contain many common IS sequences and are high in copy number. The IS sequence IS used as a homologous sequence to realize the recombination of the integration vector and the Escherichia coli genome, the integration vector can be integrated into the Escherichia coli genome through a conventional integration technology, and a metabolic pathway up to 12kb can be integrated into the genome under the assistance of lambda-Red, so that the method IS simple and easy to implement, and the target gene integrated onto the genome IS stable in heredity.
Common IS sequences in Escherichia coli include IS1, IS2, IS3, IS4, IS5 and the like, different Escherichia coli contain different IS sequence copy numbers, the lengths of homologous sequences are different, the more the copy number IS, the longer the homologous sequence IS, the more the copy number of target gene integration IS, the higher the probability IS, in the IS sequences, the copy number of IS5 IS about 12, and the homologous sequence IS 1000-1017 bp. Thus, preferably, the IS5 sequence IS selected as the integration site to increase the copy number of the gene of interest.
The integration vector uses an R6K replicon, which can replicate and amplify only in a host bacterium expressing gamma protein and can grow on a resistant culture medium only by integrating into the genome in a common strain; preferably, therefore, the replicon is an R6K replicon.
Other structures in the integration vector, such as promoters, terminators and RBS sites, can be selected according to the type of gene of interest; the type of resistance gene can also be determined according to the purpose of the study. Specifically, preferably, the base sequence of the integration vector is shown in SEQ ID NO. 1.
The invention also provides a genetic engineering bacterium, which comprises host cell escherichia coli and the integrated vector transferred into escherichia coli genome. The Escherichia coli is preferably W3110. The invention further provides the application of the genetic engineering bacteria in the synthesis of carotenoid. The target gene integrated in the genome of the engineering bacterium is the MEP pathway speed limiting step and lycopene synthesis related gene.
The promoter of the lycopene synthesis related gene is screened, and experiments show that aiming at different modules, a dxs-dxr module in a speed limiting step in an MEP pathway is under a phoR promoter, an idi-crtE module is under a yejG promoter, the lycopene synthesis gene is under a trc promoter, and the yield of lycopene is highest.
In the Escherichia coli genome of the genetic engineering bacteria, the integration vector IS integrated at the site of the Escherichia coli genome IS5 in a fixed point manner by means of the assistance of a lambda-Red recombination system and Campbell-type recombination; and after the integration is finished, redundant sequences including the resistance gene are deleted.
Compared with the prior art, the invention has the advantages that:
(1) the invention uses IS sequence in the Escherichia coli genome as integration site, integrates the vector construction and the genome, and utilizes Campbell-type recombination to integrate large segments with length of 12kb into the genome with the assistance of lambda-Red recombinant protein, thereby not only having simple integration method, but also having stable target gene heredity integrated on the genome, and simplifying the time-consuming and labor-consuming multiple rounds of integration of the existing integration technology.
(2) After the target gene in the genetically engineered bacterium is integrated into a genome, the problem of unstable plasmid separation does not exist, and after the integration is finished, redundant sequences (including resistance genes) can be deleted by means of a CRISPR/Cas9 system, so that the next round of gene operation is facilitated.
Drawings
FIG. 1 is a flow chart of the integrated technology operation.
FIG. 2 IS a map of genome integration plasmid pRC-IS 5.
FIG. 3 shows the results of different module promoter screens. a is the screening result of the lycopene synthesis module promoter, wherein trc-strain EC101, yciG-strain EC102, pstA-strain EC103, yodA-strain EC104, astC-strain EC105 and ybiM-strain EC 106; b is the screening result of a feedforward control module dxs-dxr promoter, wherein the phnI-strain EC201, the phoR-strain EC202, the phnF-strain EC203, the phnC-strain EC204 and the phnD-strain EC205 are selected; c is the result of the selection of the feedback control module idi-crtE promoter, where yfiL-strain EC301, yijF-strain EC302, cysP-strain EC303, yejG-strain EC304, yhcN-strain EC 305.
FIG. 4 shows the result of integration of large fragment genome. a is a schematic diagram of a target exogenous metabolic pathway; b is the number of colonies obtained after integration and the integration efficiency, - λ -Red: no recombinant protein, + λ -Red: contains recombinant protein; c is colony PCR validation, wherein M: DNA marker, lanes 1-6: performing colony PCR verification for single colony picked from the screening plate; d IS the comparison of the yield of lycopene by the engineering bacteria EC101 and EC-IS 5.
Fig. 5 shows the redundant sequence deletion result. a is colony PCR verification result, wherein M: DNA marker; CK, colonies not subjected to redundant sequence deletion; lanes 1-9, single colony on the plate after redundant sequence deletion by CRISPR/Cas9 system, colony PCR validation was performed, and lanes 3-5 are false positive strains, showing that the redundant sequence of this colony was not successfully deleted; b IS the comparison of the lycopene production of the engineering bacteria EC401 and EC-IS5 (delta Cm).
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples. Unless otherwise specified, the techniques used in the following examples are conventional techniques known to those skilled in the art, and the instruments, reagents and the like used are available to those skilled in the art from public sources such as commercial sources and the like.
The invention adopts the genome integration technology to integrate the target gene into the host cell genome; however, the integration of a target gene of a small DNA fragment in a genome can only be realized by adopting the existing genome integration technology, and the assembly of a metabolic pathway in Escherichia coli IS seriously influenced, so in order to solve the problems, the invention adopts a specific homologous sequence IS sequence which IS a normal composition of a host cell and has a plurality of copy numbers, has no specific function on the growth of the cell, only increases the variation probability during the cell inheritance, IS beneficial to the evolution of organisms, but IS not beneficial to industrial production. One-step integration of up to 12kb of metabolic pathways into the E.coli genome is achieved with the aid of the lambda-Red system.
The standard lycopene assay references in the following examples are: xie W, Ye L, Lv X, Xu H, YuH, Sequential control of biochemical pathways for basic evaluation of metabolic intermediates in Saccharomyces cerevisiae Eng 2015,28:8-18.
Example 1 construction of Large fragment genome integration technology System
The genome integration process IS shown in FIG. 1, and an integration plasmid containing the IS5 sequence, R6K replicon, chloramphenicol resistance gene, the desired exogenous metabolic pathway, and N20 recognition site IS first constructed (FIG. 2). The system is mainly assisted by plasmids expressing lambda-Red recombinant protein and CRISPR/Cas9 system. The integrated plasmid was integrated into the E.coli genome by Campbell-type recombination with the aid of a lambda-Red recombinant protein. And then, redundant sequences such as an IS5 sequence, an R6K replicon, a chloramphenicol resistance gene and the like are deleted under the mediation of CRISPR/Cas 9.
Example 2 different Module promoter selection
1. Lycopene synthesis pathway promoter screening
This example first uses the pET-30a-trc vector (Su B, Zhang Z, Wu M, Lin J, YangL: Construction of plasmid-free Escherichia coli for the production of antibody-free xylene from corn microbiological hydrolytical hydrochloride. Sci Rep 2016,6:26567.) deposited in the laboratory as a template, the plasmid module is amplified by the primers trc-30a-P1/trc-30a-P2, the lycopene synthetic genes crtI, crtE and crtB are respectively amplified by primers crtI-P1/crtI-P2, crtE-P1/crtE-P2 and crtB-P1/crtB-P2 (the gene sequences of the crtI, the crtE and the crtB are disclosed in Chinese patent application with the patent application number of CN 109943492A and the invention name of a recombinant yeast strain and the application thereof), and a carrier pET-trc-IEB is constructed by utilizing a recombinant kit. Subsequently, the plasmid backbone was amplified using as a template pCDFDuet-1(Su B, Zhang Z, Wu M, Lin J, Yang L: Construction of plasmid-free Escherichia coli for the production of antibody-free xylene from corn biochemical hydrosylate. Sci Rep 2016,6:26567.) deposited in the laboratory, and the primers CDF-P1/CDF-P2; using pET-trc-IEB as a template, and amplifying an IEB module by using a primer I-E-B-P1/I-E-B-P2; using Escherichia coli W3110 genome as template, using primers yodA-P1/yodA-P2, astC-P1/astC-P2, pstA-P1/pstA-P2, yciG-P1/yciG-P2, ybiM-P1/ybiM-P2 to respectively amplify yodA, astC, pstA, yciG and ybiM promoters; recombinant plasmids pCDF-yciG-IEB, pCDF-pstA-IEB, pCDF-yodA-IEB, pCDF-astC-IEB and pCDF-ybiM-IEB are respectively constructed by using the recombinant kit. The recombinant plasmids were introduced into W3110 strain to obtain recombinant strains EC101(pET-trc-IEB), EC102(pCDF-yciG-IEB), EC103(pCDF-pstA-IEB), EC104(pCDF-yodA-IEB), EC105(pCDF-astC-IEB), and EC106 (pCDF-ybiM-IEB).
The content of lycopene expressed by the above-constructed strains EC101, EC102, EC103, EC104, EC105 and EC106 was determined according to standard lycopene assay methods. The lycopene production of each strain is shown in FIG. 3 a. As can be seen from FIG. 3a, the exogenous lycopene synthesis module is under the trc promoter, with the highest yield; followed by yciG.
The primers are as follows:
and (3) PCR reaction system: the PCR reaction system was 50. mu.L, where PrimerSTAR MaxDNA Polymerase was 25. mu.L, the upstream and downstream primers (10. mu.M) were 1.5. mu.L each, the template (50ng/ml) was 1. mu.L, ddH2O 21μL；
The PCR reaction program is: pre-denaturation at 95 ℃ for 2 min; denaturation at 98 ℃ for 10s, Tm annealing for 15s, extension at 72 ℃ for 5s/kb, 30 cycles; extending for 5min at 72 ℃, and keeping the temperature at 4 ℃.
2. Feedforward control module promoter screening
This example first amplified the plasmid module with the primers pACYC-P1/pACYC-P2 using the laboratory-deposited pACYC-Duet vector (purchased from Novagen) as a template; using pCDF-yciG-IEB as a template, and amplifying a yciG-IEB module by using a primer yciG-IEB-P1/yciG-IEB-P2; dxs (Gene ID:938609) and dxr Gene (Gene ID:939636) are respectively amplified by taking a Bacillus subtilis 168(Bacillus subtilis 168) genome as a template and primers dxs-P1/dxs-P2, dxr-P1/dxr-P2; respectively amplifying phnC, phnD, phnF, phnI and phoR promoters by using Escherichia coli W3110 genome as a template and primers phnC-P1/phnC-P2, phnD-P1/phnD-P2, phnF-P1/phnF-P2, phnI-P1/phnI-P2 and phoR-P1/phoR-P2; recombinant plasmids pACYC-phnI (pACYC-phnI-dxs-dxr-yciG-IEB), pACYC-phoR (pACYC-phoR-dxs-dxr-yciG-IEB), pACYC-phnF (pACYC-phnF-dxs-dxr-yciG-IEB), pACYC-phnC (CYC-phnC-dxs-dxr-yciG-IEB) and pACYC-phnD (pACYC-phnD-dxs-dxr-yciG-IEB) are respectively constructed by using a recombinant kit. The above recombinant plasmids were introduced into the W3110 strain to obtain recombinant strains EC201(pACYC-phnI), EC202(pACYC-phoR), EC203(pACYC-phnF), EC204(pACYC-phnC), and EC205 (pACYC-phnD).
The content of lycopene expressed by the above-constructed strains EC201, EC202, EC203, EC204 and EC205 was determined according to the standard lycopene assay method. The lycopene production of each strain is shown in FIG. 3 b. As can be seen from FIG. 3b, the dxs-dxr module is under the phoR promoter, with the highest yield.
Primers for constructing the plasmid dxs-dxr module were as follows:
and (3) PCR reaction system: the PCR reaction system was 50. mu.L, where 25. mu.L of PrimerSTAR Max DNA Polymerase, 1.5. mu.L each of upstream and downstream primers (10. mu.M), 1. mu.L of template (50ng/ml), ddH2O 21μL；
The PCR reaction program is: pre-denaturation at 95 ℃ for 2 min; denaturation at 98 ℃ for 10s, Tm annealing for 15s, extension at 72 ℃ for 5s/kb, 30 cycles; extending for 5min at 72 ℃, and keeping the temperature at 4 ℃.
3. Feedback control module promoter screening
In the embodiment, plasmid modules are amplified by using a primer pACYC-P1/pACYC-P2 by using a pACYC-Duet vector preserved in a laboratory as a template; using pCDF-yciG-IEB as a template, and amplifying a yciG-IEB module by using a primer yciG-IEB-P1/yciG-IEB-P2; amplifying the idi Gene (Gene ID:938985) by using a Bacillus subtilis genome as a template and using primers idi-P1/idi-P1; using pCDF-yciG-IEB as a template, and amplifying a crtE gene by using a primer crtE-P1/crtE-P1; using Escherichia coli W3110 genome as template, and amplifying cysP, yijF, yejG, yfiL and yhcN promoters with primers cysP 1/cysP 2, yijF-P1/yijF-P2, yejG-P1/yejG-P2, yfiL-P1/yfiL-P2 and yhcN-P1/yhcN-P2, respectively; recombinant plasmids pACYC-yfiL (pACYC-yfiL-idi-crtE-yciG-IEB), pACYC-yijF (pACYC-yijF-idi-crtE-yciG-IEB), pACYC-cysP (pACYC-cysP-idi-crtE-yciG-IEB), pACYC-yejG (pACYC-yejG-idi-crtE-yciG-IEB), and pACYC-yhcN (pACYC-yhcN-idi-crtE-yciG-IEB) were constructed, respectively, using recombinant kits. The above recombinant plasmids were introduced into the W3110 strain to obtain recombinant strains EC301(pACYC-yfiL), EC302(pACYC-yijF), EC303(pACYC-cysP), EC304(pACYC-yejG), and EC305(pACYC-yhcN), respectively.
The content of lycopene expressed by the above-constructed strains EC301, EC302, EC303, EC304 and EC305 was determined according to the standard lycopene assay method. The lycopene production of each strain is shown in FIG. 3 c. As can be seen in FIG. 3c, the idi-crtE module is under the yejG promoter with the highest yield.
Primers for constructing the plasmid idi-crtE module were as follows:
and (3) PCR reaction system: the PCR reaction system was 50. mu.L, where 25. mu.L of PrimerSTAR Max DNA Polymerase, 1.5. mu.L each of upstream and downstream primers (10. mu.M), 1. mu.L of template (50ng/ml), ddH2O 21μL；
The PCR reaction program is: pre-denaturation at 95 ℃ for 2 min; denaturation at 98 ℃ for 10s, Tm annealing for 15s, extension at 72 ℃ for 5s/kb, 30 cycles; extending for 5min at 72 ℃, and keeping the temperature at 4 ℃.
Example 3 Large fragment genomic integration
After the plasmid pACYC-phoR (pACYC-phoR-dxs-dxr-ysiG-IEB) was used as a template, the plasmid pPCR-dxs-dxr-P-3, the plasmid pPCR-dxs-dxr-P-2, the plasmid pPCR-dxs-P/phoR-dxs-dxr-P was amplified using the primers PhoCYC-yejG (pACYC-yejG-idi-crtE-yc-IEB) as a template, the plasmid pPCR-yPCR-induced colony protein was amplified using the primers yejG-idi-crtE-P/yejG-crtE-E-P, the plasmid pPCR-induced colony PCR-induced by the PCR-induced by the primers, the PCR-induced colony PCR-induced by the PCR-induced expression of the plasmid pPCR-induced colony protein (see the PCR-induced colony protein expression of the colony-expression of the plasmid pPCR-induced colony protein, the colony-induced colony-expression of the plasmid pPCR-induced colony protein, the plasmid pPCR-induced colony-induced by the PCR-induced colony protein, the plasmid pPCR-induced colony-induced plasmid, the plasmid pPCR-induced colony expression of the plasmid was observed when the plasmid was found to contain the plasmid pPCR-induced by the plasmid pPCR-induced colony protein, the plasmid was found to contain the plasmid pPCR-induced colony protein, the plasmid pPCR-induced by the plasmid, the plasmid was found to contain the plasmid pPCR-induced by the plasmid pPCR-induced colony expression of the plasmid, the plasmid was found to contain the plasmid, the plasmid was found to contain the plasmid pPCR-induced plasmid, the plasmid was not to contain the plasmid, the plasmid was found to contain the plasmid, the plasmid was found to contain the plasmid, the plasmid was found to be the plasmid pPCR-induced plasmid, the plasmid was the plasmid, the plasmid was found to be the plasmid was found to contain the plasmid was found to be the plasmid was found to contain the plasmid, the plasmid was found to be the plasmid was the plasmid, the plasmid was found to be the plasmid, the plasmid was found to be the plasmid was found to be the plasmid was found to.
The EC-IS5 strain constructed above and the EC101 strain constructed in example 2 were assayed for lycopene synthesis according to standard lycopene assay methods. The lycopene production of each strain is shown in FIG. 4 d. As can be seen from FIG. 4d, the yield of the EC-IS5 strain was increased 40-fold by genomic integration.
The PhoR-dxs-dxr module primers were amplified as follows:
| Primers | Sequence(5'-3') |
| phoR-dxs-dxr-P1-3 | aaaatctcaaaaatctcaaaaggaaggtaactcttcaaatgtagcacctgaagt (underline represents homology arm) |
| phoR-dxs-dxr-P1-2 | ctcttcaaatgtagcacctgaagtgagctcagctttattgagtgg (underline represents homology arm) |
| phoR-dxs-dxr-P1 | gagctcagctttattgagtggcctcgactggatgttacctggc (underline represents homology arm) |
| phoR-dxs-dxr-P2 | tcagcaaaaaacccctcaagacc |
Primers for amplification of the yejg-idi-crtE module were as follows:
| Primers | Sequence(5'-3') |
| yejG-idi-crtE-P1 | ggtcttgaggggttttttgctgagctggtggttggcgttgcgg (underline represents homology arm) |
| yejG-idi-crtE-P2 | caccaaacgtttcggcgagaagcttgactaccggaagcagtgtg (underline represents homology arm) |
The lacI-trc-IEB terminal module primers were amplified as follows:
| Primers | Sequence(5'-3') |
| IEB-P1 | gcttctcgccgaaacgtttggtgg |
| IEB-P2 | catgcgggacaagaaaatctcttatccggatatagttcctcctttcag (underline represents homology arm) |
The primers for amplifying the IS5-R6K-Cm module are as follows:
| Primers | Sequence(5'-3') |
| IS5-R6K-CM-P1 | aagagattttcttgtcccgcatg |
| IS5-R6K-CM-P2 | ccatgtccttagttcggcttctgcaaataaaacgaaaggctcagtcg (underlined generation)Watch arm of homology) |
| IS5-R6K-CM-P2-2 | ttaccttccttttgagatttttgagattttccatgtccttagttcggcttctg (underline represents homology arm) |
The primers were identified as follows:
| Primers | Sequence(5'-3') |
| IS5-check-P1 | aagagattttcttgtcccgcatggagcag |
| IS5-check-P2 | cagcggtagcaacagccagacgag |
and (3) PCR reaction system: the PCR reaction system was 50. mu.L, where 25. mu.L of PrimerSTAR Max DNA Polymerase, 1.5. mu.L each of upstream and downstream primers (10. mu.M), 1. mu.L of template (50ng/ml), ddH2O 21μL；
The PCR reaction program is: pre-denaturation at 95 ℃ for 2 min; denaturation at 98 ℃ for 10s, Tm annealing for 15s, extension at 72 ℃ for 5s/kb, 30 cycles; extending for 5min at 72 ℃, and keeping the temperature at 4 ℃.
Example 4 redundant sequence deletion
Plasmid pTargetF-delete IS constructed by whole plasmid PCR using primers delete-CRISPR-P1/delete-CRISPR-P2 as a template pTargetF-cadA (Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigeneanding in the Escherichia coli gene via the CRISPR-Cas9 system. ApplEnviron Microbiol 2015,81:2506-2514.), and IS introduced into engineering bacterium EC-IS5 (example 3) by electrotransformation with primers IS5-R6K-Cm-delete, and IS cultured with engineering bacterium EC-IS5 as a Control (CK) for 3 hours, after which the supernatant IS removed by centrifugation, 100. mu.L of sterile water IS added, kanamycin IS spread on spectinomycin plate for 30 ℃ and cultured at 16 ℃. Randomly picking 9 colonies on the plate, carrying out colony PCR verification by using identification primers IS5-check-P1/IS5-check-P2, obtaining the engineering bacteria EC-IS5 (delta Cm) by obtaining correct clones when the contrast of PCR products and CK becomes smaller after nucleic acid electrophoresis, and obtaining the result shown in figure 5 a.
The EC-IS5(Δ Cm) strain constructed above, and the EC401 strain constructed in example 3, were assayed for lycopene expression according to standard lycopene assay methods. The lycopene production of each strain is shown in FIG. 5 b. As can be seen from FIG. 5b, the yield of the genome-integrated strain (EC-IS5 (. DELTA.Cm)) was increased 4.4-fold as compared with the plasmid-engineered strain (EC 401).
The primers for the redundant sequences were as follows:
primers for constructing pTargetF-delete plasmid were as follows:
| Primers | Sequence(5'-3') |
| delete-CRISPR-P1 | cagaagccgaactaaggacagttttagagctagaaatagc (underline represents homology arm) |
| delete-CRISPR-P2 | tgtccttagttcggcttctgactagtattatacctaggac (underline represents homology arm) |
And (3) PCR reaction system: the PCR reaction system was 50. mu.L, where 25. mu.L of PrimerSTAR Max DNA Polymerase, 1.5. mu.L each of upstream and downstream primers (10. mu.M), 1. mu.L of template (50ng/ml), ddH2O 21μL；
The PCR reaction program is: pre-denaturation at 95 ℃ for 2 min; denaturation at 98 ℃ for 10s, Tm annealing for 15s, extension at 72 ℃ for 5s/kb, 30 cycles; extending for 5min at 72 ℃, and keeping the temperature at 4 ℃.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)
<120> genome integration method and use
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>14833
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
aaaatctcaa aaatctcaaa aggaaggtaa ctcttcaaat gtagcacctg aagtgagctc 60
agctttattg agtggcctcg actggatgtt acctggcggc tccggtatcc agttcatcaa 120
acacctcaag cgcgagtcga tgacccggga tattccagtg gtgatgttga ccgccagagg 180
ggaagaagaa gatcgcgtgc gcggccttga aaccggcgcg gatgactata tcaccaagcc 240
gttttcgccg aaggagctgg tggcgcgaat caaagcggta atgcgccgta tttcgccaat 300
ggcggtggaa gaggtgattg agatgcaggg attaagtctc gacccgacat ctcaccgagt 360
gatggcgggc gaagagccgc tggagatggg gccgacagaa tttaaactgc tgcacttttt 420
tatgacgcat cctgagcgcg tgtacagccg cgagcagctg ttaaaccacg tctggggaac 480
taacgtgtat gtggaagacc gcacggtcga tgtccacatt cgtcgcctgc gtaaagcact 540
ggagcccggc gggcatgacc gcatggtgca gaccgtgcgc ggtacaggat atcgtttttc 600
aacccgcttt taacgccttg ctcatcggac gcagagcagg gcgtttaact ttaagaagga 660
gatataccat ggatctttta tcaatacagg acccgtcgtt tttaaaaaac atgtccattg 720
atgaattaga gaaattaagt gatgaaatcc gtcagttttt aattacaagt ttatccgctt 780
ccggcggcca catcggccca aacttaggtg tcgtagagct tactgttgcc ctgcataagg 840
aatttaacag cccgaaagac aaatttttat gggatgtagg ccatcagtcg tatgtccata 900
agctgctgac aggacgcgga aaagaatttg cgacgcttcg ccagtacaaa gggctttgcg 960
gatttccaaa gcggagtgaa agcgagcacg atgtttggga aaccgggcac agctcgactt 1020
ctctgtcagg cgcgatggga atggcagctg cccgtgatat taaaggaacg gatgaatata 1080
ttattccgat cattggtgac ggcgcgctga ccggcggtat ggcgctcgaa gcccttaacc 1140
acatcggcga cgagaaaaaa gacatgattg tcatccttaa tgataatgaa atgagtattg 1200
cgccaaacgt cggtgccatt cactctatgc tcggacggct ccgcactgcg gggaaatacc 1260
agtgggtcaa agatgagctt gaatacttat ttaaaaagat tccggcagtt gggggcaagc 1320
ttgccgccac ggcggaacgg gtcaaagaca gcctgaaata catgctcgtc tccggaatgt 1380
ttttcgagga gctcggtttt acgtatttgg gcccagtgga cggacattct tatcatgagc 1440
tgattgagaa tcttcaatac gccaaaaaaa cgaaaggccc tgttcttctg cacgtcatta 1500
cgaaaaaagg gaaggggtac aaaccggctg agaccgatac gattgggaca tggcatggta 1560
ccggaccata taaaattaat accggtgact ttgtaaagcc gaaagccgca gctccttcgt 1620
ggagcggtct tgtcagcgga actgtgcagc gaatggcgcg cgaggacgga cgcattgtag 1680
ccattacgcc ggctatgcct gtcggttcaa agcttgaagg cttcgcaaag gaattccctg 1740
accggatgtt cgacgtagga atcgcagaac agcatgccgc aacaatggct gcagctatgg 1800
caatgcaggg tatgaagccg tttttggcga tttactcaac cttcctgcaa agggcatatg 1860
accaagttgt tcatgacatc tgccgccaaa acgctaatgt gtttattgga attgaccgtg 1920
ctggactcgt tggcgctgat ggagagacac atcaaggcgt gtttgatatt gcgtttatgc 1980
gccacattcc aaacatggtc ttaatgatgc cgaaagacga aaatgaaggc cagcacatgg 2040
ttcatacagc acttagctat gacgaaggcc cgatagcaat gcgttttccg cgcggaaacg 2100
gactcggcgt aaaaatggat gaacagttga aaacgattcc gatcggtacg tgggaggtgc 2160
tgcgtccagg gaacgatgct gtcatcttaa cattcggcac aacaatcgaa atggcgattg 2220
aagcagccga agagctgcag aaagaaggcc tttccgtgcg cgttgtgaat gcgcgtttta 2280
ttaagccgat tgatgaaaag atgatgaaga gtatcctaaa agaaggcttg ccaattttaa 2340
caattgaaga agcggtctta gaaggcggtt tcggaagctc gattttagaa ttcgctcatg 2400
atcaaggtga atatcatact ccgattgaca gaatgggtat acctgatcgg tttattgaac 2460
acggaagtgt aacagcgctt cttgaggaaa ttggactgac aaaacagcag gtggcaaatc 2520
gtattagatt actgatgcca ccaaagacac acaaaggaat tggatcatga tttaataagg 2580
aggaaaacaa atgaaaaata tttgtctttt aggagcaaca ggatcaatcg gcgaacagac 2640
tcttgatgta ctgcgtgcac atcaagacca atttcagctg gtatctatgt cgtttggcag 2700
aaatattgat aaggccgttc caatgattga ggtctttcag ccgaagtttg tctctgtcgg 2760
tgatctggat acatatcata aattaaaaca aatgtctttt tcttttgaat gccaaatcgg 2820
gctgggagaa gaaggactga tagaagcagc agtgatggag gaggttgaca ttgttgtcaa 2880
cgctttgctt ggaagcgtcg gtcttatccc gacattaaag gcaattgaac agaaaaaaac 2940
aattgcgctt gcaaataagg aaactcttgt cactgcaggg catatagtaa aagaacatgc 3000
taagaaatat gatgttccgc tgctgcctgt tgacagtgag cattcagcca tttttcaagc 3060
tcttcaaggc gaacaggcta aaaacattga acgcctcatc attacggcat ccggcggaag 3120
ttttcgggac aagacgcggg aggagctcga atcagtaacg gttgaagatg cattaaaaca 3180
tccaaactgg tcaatgggtg caaaaattac aattgattcg gctacaatga tgaataaggg 3240
attagaggtg atcgaggcac actggctttt cgatatacct tatgaacaaa ttgatgtggt 3300
tttacataag gagagcatca tccattcaat ggttgagttc catgataaaa gtgtgatcgc 3360
acagctcgga actccggata tgagagtccc cattcaatat gcgctcactt atcctgaccg 3420
attgccatta ccagatgcga aaaggcttga attatgggaa atcggcagcc ttcattttga 3480
gaaagctgat tttgacaggt tccgatgctt acaatttgct tttgaatcag gtaaaatagg 3540
aggaacaatg ccgacagtgc taaatgcggc aaacgaagta gctgtcgctg cctttttagc 3600
tggcaagata ccgtttttgg ctattgaaga ctgtatcgaa aaggcactaa cccgccatca 3660
attactgaaa aaaccgagcc tggcggacat tcaagaagtg gacaaagata cccggggata 3720
cgtcaattca atactcacat aaaataacta gcataacccc ttggggcctc taaacgggtc 3780
ttgaggggtt ttttgctgag ctggtggttg gcgttgcggc gtttgtgggc tgcgtgtcga 3840
tggtgtcatc caatgcgatg gcggtcattc ttgatgagtt tccccatatg gcgggaacgg 3900
catcttcgct ggcaggaacc ttccgttttg gcataggggc aattgttggc gcattgcttt 3960
ctcttgcgac ctttaactct gcatggccga tgatttggtc aattgcattc tgcgcaacca 4020
gctccattct cttctgtctg tacgccagtc ggccgaaaaa acggtgatct attgcacaac 4080
gaggaagcta aaaggcttcc tttgttgatg catgtcaacc acaaatctat cattcccccg 4140
atatatgttt attttatgta aaatcaattt atgtaaaaag tcacatcatt gtagttaaaa 4200
aggttgagtt agatcgcaga aacgggtaca tatagccccg caaacgtgac cacgcccgca 4260
gatattactt aaatcagagc catagaggcc acgcaggcga ggcatcaatc tttacgatct 4320
gtataaagac ggattgttga tgatgtgtta aaattgatga aatacggaac aaggaggaaa 4380
acaaatgact cgagcagaac gaaaaagaca acacatcaat catgccttgt ccatcggcca 4440
gaagcgggaa acaggtcttg atgatattac gtttgttcac gtcagtctgc ccgatcttgc 4500
attagaacaa gtagatattt ccacaaaaat cggcgaactt tcaagcagtt cgccgatttt 4560
tatcaatgca atgactggcg gcggcggaaa acttacatat gagattaata aatcgcttgc 4620
gcgagcggct tctcaggctg gaattcccct tgctgtggga tcgcaaatgt cagcattaaa 4680
agatccatca gagcgtcttt cctatgaaat tgttcgaaag gaaaacccaa acgggctgat 4740
ttttgccaac ctgggaagcg aggcaacggc tgctcaggca aaggaagccg ttgagatgat 4800
tggagcaaac gcactgcaga tccacctcaa tgtgattcag gaaattgtga tgcctgaagg 4860
ggacagaagc tttagcggcg cattgaaacg cattgaacaa atttgcagcc gggtcagtgt 4920
accggtcatt gtgaaagaag tcggcttcgg tatgagcaaa gcatcagcag gaaagctgta 4980
tgaagctggt gctgcagctg ttgacattgg cggttacggg ggaacaaatt tctcgaaaat 5040
cgaaaatctc cgaagacagc ggcaaatctc cttttttaat tcgtggggca tttcgacagc 5100
tgcaagtttg gcggaaatcc gctctgagtt tcctgcaagc accatgatcg cctctggcgg 5160
tctgcaagat gcgcttgacg tggcaaaggc aattgcgctg ggggcctctt gcaccggaat 5220
ggcagggcat tttttaaaag cgctgactga cagcggtgag gaaggactgc ttgaggagat 5280
tcagctgatc cttgaggaat taaagttgat tatgaccgtg ctgggtgcca gaacaattgc 5340
cgatttacaa aaggcgcccc ttgtgatcaa aggtgaaacc catcattggc tcacagagag 5400
aggggtcaat acatcaagct atagtgtgcg ataaaagaag gagatataca tatgcgtccg 5460
gaactgctgg aacgtgttct gtctctgctg ccggaagctg gtccgcaccc ggaactggct 5520
cgtttctacg aaatgctgcg tgactacccg cgtcgtggtg gtaaaggtct gcgttctgaa 5580
ctgctgctgg ctggtgctcg tgcttacggt gttcgtgaag gtaccccgca gtgggaatct 5640
gctctgtggc tggctgctgg tgttgaactg ttccagaact gggttctgat ccacgacgac 5700
atcgaagacg actctgaaga acgtcgtggt cgtccggctc tgcaccgtct gcacggtgtt 5760
gctctggcta tcaacgctgg tgacgctctg cacgcttaca tgtgggctgc tgttgctcgt 5820
gctggtgttc cgggtgctca cgaagaattc ctggctatgg ttcaccgtac cgctgaaggt 5880
cagcacctgg acctggcttg ggttgctggt cgtgaatggg gtctgaccga acacgactac 5940
ctgcagatgg ttggtctgaa aaccgcttac tacaccgtta tcgttccgct gcgtctgggt 6000
gctctggttg ctggtgctca gccgccggaa accctgaccc cggctggtct ggctctgggt 6060
accgctttcc agatccgtga cgacgttctg aacctggctg gtgacgctgc taaatacggt 6120
aaagaaatcg ctggtgacct gctggaaggt aaacgtaccc tgatcgttct gcactggctg 6180
ggtcaggctc cggaagacca gatcgctgtt ttcctggacc agatgcgtcg tgaacgtccg 6240
gacaaagacc cggaagttgt tgctcagatc cacggttggc tgctggaatc tggttctgtt 6300
gactacgctc agcgtgctgc tcaggctcag gctgaaaccg gtctgaaact gctgggtgac 6360
gttctgggtg ctgctccgga acgtgaagct gctcgtgctc tgctgggtcg tgttcgtgaa 6420
ctggctaccc gtgaagctta aaataactag cataacccct tggggcctct aaacgggtct 6480
tgaggggttt tttgctgaaa cctcaggcat ttgagaagca cacggtcaca ctgcttccgg 6540
tagtcaagct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg 6600
gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag 6660
cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata 6720
aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg 6780
actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa 6840
cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 6900
ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt 6960
ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg 7020
agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat 7080
ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga 7140
gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat 7200
ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt 7260
ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg 7320
attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa 7380
tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag 7440
tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc 7500
aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc 7560
cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc 7620
tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg 7680
atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga 7740
ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg 7800
aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg 7860
gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac 7920
atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta 7980
tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc 8040
ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg 8100
ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc 8160
ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt 8220
ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc 8280
cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg aaatgttgac 8340
aattaatcat ccggctcgta taatgtgtgg aattgtgagc ggataacaat tcccctctag 8400
aaataatttt gtttaacttt aagaaggaga tatacatatg accgtttctt ctgcttctcc 8460
gcgtccggct ggtccgccgc agcgtaaatc tgctctgatc gttggtgctg gtatcggtgg 8520
tctgtctctg ggtatccgtc tgcagtctct gggtttcgac accaccatcc tggaacgtct 8580
ggacggtccg ggtggtcgtg cttaccagaa acgtaccgaa gacggttacg ttttcgacat 8640
gggtccgacc gttatcaccg ttccgcactt catcgaagaa ctgttctctc tggaacgtga 8700
ccacgctgct ctgaacaccc cggactaccc gccgcacacc ctgtctggtg aacgtgttaa 8760
agctggtgac tctggtggtc cgcgtacccg tgaatacgtt aacctggttc cgatcctgcc 8820
gttctaccgt atcgttttcg acgacgctac cttcttcgac tacgacggtg acccggtttc 8880
tacccgtgaa cagatcgctc gtctggctcc ggaagacctg gaaggttacg aacgtttcca 8940
ccgtgacgct caggctatct tcgaacgtgg tttcctggaa ctgggttaca cccacttcgg 9000
tgacctgccg accatgctgc gtgttgttcc ggacctgatg aaactggacg ctgttcgtac 9060
cctgttctct ttcacctctc gttacttctc ttctgacaaa atgcgtcagg ttttctcttt 9120
cgaaaccctg ctgatcggtg gtaacccgct gtctgttccg gctatctacg ctatgatcca 9180
cttcgttgaa aaaacctggg gtgttcacta cgctatgggt ggtaccggtg ctctggttca 9240
gggtttcgtt cgtaaattcc gtgaactggg tggtaccgtt cgttacggta ccggtgttga 9300
agaaatcctg gttgaatctg gtcgtggtgg tccggttcgt gctccggttg gtccgcgtgt 9360
tgctcgtggt gttcgtctgg aatctggtga agaactgcgt gctgacatcg ttgtttctaa 9420
cggtgactgg gctaacacct acctgaaacg tgttccggct gctgctcgtc tggttaacaa 9480
cgacctgcgt atcaaagctg ctccgcagtc tatgggtctg ctggttatct acttcggttt 9540
ccgtgacgac ggtcagccgc tgaacctgcg tcaccacaac atcctgctgg gtccgcgtta 9600
cgaagctctg ctgcgtgaaa tcttcggtaa aaaagttctg ggtcaggact tctctcagta 9660
cctgcacgtt ccgaccctga ccgacccggc tctggctccg gctggtcacc acgctgctta 9720
caccctggtt ccggttccgc acaacggttc tggtatcgac tggtctgttg aaggtccgcg 9780
tctgaccgaa cgtgttctgg actacctgga agaacgtggt ttcatcccgg acctgcgtgc 9840
tcgtctgacc cacttcgaat acgttacccc ggactacttc gaaggtaccc tggactctta 9900
cctgggtaac gctttcggtc cggaaccggt tctggctcag tctgctttct tccgtccgca 9960
caaccgttct gaagacgttc gtggtctgta cctggttggt gctggtgctc agccgggtgc 10020
tggtaccccg tctgttatga tgtctgctaa aatgaccgct cgtctgatcg ctgaagactt 10080
cggtatccac ccggacctgc tgggtgctgc tgctgacgac taaaatacgg aacaaggagg 10140
aaaacaaatg cgtccggaac tgctggaacg tgttctgtct ctgctgccgg aagctggtcc 10200
gcacccggaa ctggctcgtt tctacgaaat gctgcgtgac tacccgcgtc gtggtggtaa 10260
aggtctgcgt tctgaactgc tgctggctgg tgctcgtgct tacggtgttc gtgaaggtac 10320
cccgcagtgg gaatctgctc tgtggctggc tgctggtgtt gaactgttcc agaactgggt 10380
tctgatccac gacgacatcg aagacgactc tgaagaacgt cgtggtcgtc cggctctgca 10440
ccgtctgcac ggtgttgctc tggctatcaa cgctggtgac gctctgcacg cttacatgtg 10500
ggctgctgtt gctcgtgctg gtgttccggg tgctcacgaa gaattcctgg ctatggttca 10560
ccgtaccgct gaaggtcagc acctggacct ggcttgggtt gctggtcgtg aatggggtct 10620
gaccgaacac gactacctgc agatggttgg tctgaaaacc gcttactaca ccgttatcgt 10680
tccgctgcgt ctgggtgctc tggttgctgg tgctcagccg ccggaaaccc tgaccccggc 10740
tggtctggct ctgggtaccg ctttccagat ccgtgacgac gttctgaacc tggctggtga 10800
cgctgctaaa tacggtaaag aaatcgctgg tgacctgctg gaaggtaaac gtaccctgat 10860
cgttctgcac tggctgggtc aggctccgga agaccagatc gctgttttcc tggaccagat 10920
gcgtcgtgaa cgtccggaca aagacccgga agttgttgct cagatccacg gttggctgct 10980
ggaatctggt tctgttgact acgctcagcg tgctgctcag gctcaggctg aaaccggtct 11040
gaaactgctg ggtgacgttc tgggtgctgc tccggaacgt gaagctgctc gtgctctgct 11100
gggtcgtgtt cgtgaactgg ctacccgtga agcttaatta actttaagaa ggagatatac 11160
catgaccgac tgcgctccgc cggctccgtc ttctaccgtt gacccgggtc tgaaccgtgc 11220
tctgcgtcac tgccaggctg ttacccgtga acactctaaa accttctacc tgggttctcg 11280
ttgcttcccg ggtcgtcagc gtgctgctgt ttgggctgtt tacgctgctt gccgtgaagg 11340
tgacgacatc gctgacggtg gtggtccgga cgttgacgct cgtctgggtg actggtggtc 11400
tcgtgttcag ggtgctttcg ctggtcgtcc gggtgaacac ccgaccgacc gtgctctggc 11460
ttgggctgct cgtgaatacc cgatcccgct gggtgctttc gctgaactgc acgaaggtct 11520
gcgtatggac ctgcgtggtc acaactacgc ttctatggac gacctgaccc tgtactgccg 11580
tcgtgttgct ggtgttgttg gtttcatgat cgctccgatc tctggttacg aaggtggtga 11640
agctaccctg gacaaagctc tgcgtctggg tcaggctatg cagctgacca acatcctgcg 11700
tgacgttggt gaagacctgt ctctgggtcg tgtttacctg ccggctgaag ttctggaccg 11760
ttacggtctg tgccgtgctg acctggaacg tggtgttgtt accccggaat actgcgctat 11820
gctgcgtgac ctgaccgctc aggctcgtgc ttggtacgct gaaggtcgtg ctggtatccc 11880
gctgctgcgt ggtcgtgctc gtctggctgt tgctaccgct gctcgtgctt acgaaggtat 11940
cctggacgac ctggaagctg ctggttacga caacttcaac cgtcgtgctt acgtttctgg 12000
tcgtcgtaaa ctgatgatgc tgccgcaggc ttggtgggaa ctgcgttctt tctctgctta 12060
aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag 12120
gaggaactat atccggataa gagattttct tgtcccgcat ggagcagatt ctgccatggc 12180
aaaacatggt ggaagtcatc gagccgtttt accccaaggc tggtaatggc cggcgacctt 12240
atccgctgga aaccatgcta cgcattcact gcatgcagca ttggtacaac ctgagcgatg 12300
gcgcgatgga agatgctctg tacgaaatcg cctccatgcg tctgtttgcc cggttatccc 12360
tggatagcgc cttgccggac cgcaccacca tcatgaattt ccgccacctg ctggagcagc 12420
atcaactggc ccgccaattg ttcaagacca tcaatcgctg gctggccgaa gcaggcgtca 12480
tgatgactca aggcaccttg gtcgatgcca ccatcattga ggcacccagc tcgaccaaga 12540
acaaagagca gcaacgcgat ccggagatgc atcagaccaa gaaaggcaat cagtggcact 12600
ttggcatgaa ggcccacatt ggtgtcgatg ccaagagtgg cctgacccac agcctggtca 12660
ccaccgcggc caacgagcat gacctcaatc agctgggtaa tctgctgcat ggagaggagc 12720
aatttgtctc agccgatgcc ggctaccaag gggcgccaca gcgcgaggag ctggccgagg 12780
tggatgtgga ctggctgatc gccgagcgcc ccggcaaggt aagaaccttg aaacagcatc 12840
cacgcaagaa caaaacggcc atcaacatcg aatacatgaa agccagcatc cgggccaggg 12900
tggagcaccc atttcgcatc atcaagcgac agttcggctt cgtgaaagcc agatacaagg 12960
ggttgctgaa aaacgataac caactggcga tgttattcac gctggccaac ctgtttcggg 13020
cggaccaaat gatacgtcag tgggagagat ctcactaagt aactgtcaga ccaagtttac 13080
tcatatatac tttagattga tttaaaactt catttttaat ttttgcggcc gcaagatccg 13140
cagttcaacc tgttgatagt acgtactaag ctctcatgtt tcacgtacta agctctcatg 13200
tttaacgtac taagctctca tgtttaacga actaaaccct catggctaac gtactaagct 13260
ctcatggcta acgtactaag ctctcatgtt tcacgtacta agctctcatg tttgaacaat 13320
aaaattaata taaatcagca acttaaatag cctctaaggt tttaagtttt ataagaaaaa 13380
aaagaatata taaggctttt aaagctttta aggtttaacg gttgtggaca acaagccagg 13440
gatgtaacgc actgagaagc ccttagagcc tctcaaagca attttcagtg acacaggaac 13500
acttaacggc tgacatggga attagccatg gtccatatga atatcctcct tagttcctat 13560
tccgaagttc ctattctcta gaaagtatag gaacttcggc gcgcctacct gtgacggaag 13620
atcacttcgc agaataaata aatcctggtg tccctgttga taccgggaag ccctgggcca 13680
acttttggcg aaaatgagac gttgatcggc acgtaagagg ttccaacttt caccataatg 13740
aaataagatc actaccgggc gtattttttg agttgtcgag attttcagga gctaaggaag 13800
ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta 13860
aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc 13920
tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct 13980
ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attacgtatg gcaatgaaag 14040
acggtgagct ggtgatatgg gatagtgttc acccttgttacaccgttttc catgagcaaa 14100
ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca 14160
tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta 14220
ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa 14280
acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc 14340
aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct 14400
tccatgtcgg cagatgctta atgaatacaa cagtactgcg atgagtggca gggcggggcg 14460
taaggcgcgc catttaaatg aagttcctat tccgaagttc ctattctcta gaaagtatag 14520
gaacttcgaa gcagctccag cctacacaat cgctcaagac gtgtaatgct gcaatctgca 14580
tgcaagcttg gcactggcca cgcaaaaagg ccatccgtca ggatggcctt ctgcttaatt 14640
tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg ttgcttcgca 14700
acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa 14760
cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg cagaagccga 14820
actaaggaca tgg 14833
Claims (10)
1. An integration vector of an escherichia coli genome comprises a replicon, a target exogenous metabolic pathway, a resistance gene and an integration site, and is characterized in that the target exogenous metabolic pathway comprises a rate-limiting step in an MEP pathway and a lycopene synthesis gene.
2. The integration vector of claim 1, wherein the rate-limiting step in the MEP pathway comprises a feed forward control module dxs-dxr and a feedback control module idi-crtE, wherein the dxs-dxr module is under the phoR promoter and the idi-crtE module is under the yejG promoter.
3. The integration vector of claim 1 or 2, wherein the lycopene synthesis gene is under the trc promoter.
4. The integration vector of claim 3, wherein the lycopene synthesis gene comprises crtE, crtB and crtI genes.
5. The integration vector of claim 1, wherein the integration site IS an IS sequence; the replicon is R6K; the resistance gene is a chloramphenicol resistance gene.
6. The integration vector of claim 5, wherein the IS sequence IS an IS5 sequence.
7. The integration vector of claim 1, wherein the base sequence of the integration vector is represented by SEQ id No. 1.
8. A genetically engineered bacterium comprising Escherichia coli and an integration vector transferred into Escherichia coli genome, wherein the integration vector is according to any one of claims 1 to 7.
9. The genetically engineered bacterium of claim 8, wherein the site-specific integration of the integration vector of any one of claims 1 to 7 into the E.coli genome IS at the IS5 site by Campbell-type recombination with the aid of a lambda-Red recombination system; and after the integration is finished, redundant sequences including the resistance gene are deleted.
10. Use of the genetically engineered bacterium of claim 8 or 9 for the synthesis of carotenoids.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773729A (en) * | 2012-10-22 | 2014-05-07 | 中国科学院上海生命科学研究院 | Recomposed escherichia coli base cell for efficient synthesis of terpene chemical compounds as well as preparation method and application thereof |
| CN104789586A (en) * | 2015-04-23 | 2015-07-22 | 浙江大学 | Escherichia coli genome integration vector, genetically engineered bacterium and application of genetically engineered bacterium to xylitol production |
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2020
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773729A (en) * | 2012-10-22 | 2014-05-07 | 中国科学院上海生命科学研究院 | Recomposed escherichia coli base cell for efficient synthesis of terpene chemical compounds as well as preparation method and application thereof |
| CN104789586A (en) * | 2015-04-23 | 2015-07-22 | 浙江大学 | Escherichia coli genome integration vector, genetically engineered bacterium and application of genetically engineered bacterium to xylitol production |
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| XIXIAN CHEN ET AL.: "Integrating Enzyme and Metabolic Engineering Tools for Enhanced α‑Ionone Production", 《J. AGRIC. FOOD CHEM》 * |
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Address after: No. 56, courtyard, No. 100, Xianlie Middle Road, Guangzhou, Guangdong 510070 Patentee after: Institute of Microbiology, Guangdong Academy of Sciences Country or region after: China Address before: 510070 No.56 courtyard, No.100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province Patentee before: GUANGDONG INSTITUTE OF MICROBIOLOGY (GUANGDONG DETECTION CENTER OF MICROBIOLOGY) Country or region before: China |