CN110791468A - Construction method and application of mycobacterium genetic engineering bacteria - Google Patents
Construction method and application of mycobacterium genetic engineering bacteria Download PDFInfo
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Abstract
The invention discloses a construction method and application of a mycobacterium genetic engineering bacterium, belonging to the technical field of genetic engineering. After the recombinant plasmid is transformed into a mycobacterium LY-1 cell, sgRNA can recognize a specific region on a genome and guide Cas9 protein to be combined to a target site, and under the action of the protein, a gap of double-strand break is formed at the target site, and most cells die: the homologous repair sequence introduced with the recombinant plasmid is integrated to the gap through double exchange under the action of recombinase, so that cells successfully repaired by gene homology survive, and artificially designed genotypes such as gene inactivation, foreign gene insertion and the like are formed. The CRISPR-Cas9 system constructed by the invention has the knockout efficiency of 60% aiming at the same gene, and is more convenient and efficient compared with the existing mycobacteria gene editing mode.
Description
Technical Field
The invention relates to a construction method and application of a mycobacterium genetic engineering bacterium, belonging to the technical field of genetic engineering.
Background
Steroid drugs have important physiological activities, are widely used for clinically treating rheumatism, cardiovascular diseases, collagenous diseases, lympholeukemia, organ transplantation of human bodies, tumor resistance, bacterial encephalitis, skin diseases, endocrine dyscrasia, senile diseases and the like, are second class drugs which are only second class drugs to antibiotics, currently, the annual sales of the steroid drugs worldwide exceed 1000 hundred million dollars, 9 α -hydroxyandrost-4-ene-3, 17-dione (9 α -OH-AD) is used as an important precursor of the steroid hormones, and a special α -configuration hydroxyl group in the structure is an important precursor for obtaining halogenated corticosteroids, such as dexamethasone, betamethasone, mometasone furoate and the like, and has important commercial value and wide market demand.
The microbial fermentation production of sterol substances can simplify the process route and reduce the production cost, and the mycobacterium is an important microorganism for producing 9 α -OH-AD by degrading sterol substances in the industry, wherein the mycobacterium comprises a complex catalytic enzyme system, 9 α -OH-AD is obtained by the ninth position hydroxylation of Androstenedione (AD), and simultaneously AD and 9 α -OH-AD can be both subjected to 3-sterone-delta-AD1Dehydrogenase (3-ketosteroid-. DELTA.1The-dehydrogenase) and the 17 β -hydroxysteroid dehydrogenase (17 β -hydroxyandrostehydrogenase) are respectively degraded into Androstenedione (ADD), 9 α -hydroxy-androstenedione (9 α -OH-ADD), testosterone (T) and 9 α -hydroxytestosterone (9 α -OH-T), and due to a complex enzyme catalytic system, on one hand, not only 9 α -OH-AD and a plurality of byproducts are generated in the sterol metabolism process, but also the molar yield of the target product 9 α -OH-AD is reduced.
The CRISPR/Cas technology is a fourth method which can be used for constructing genes at fixed points after Zinc Finger Nuclease (ZFN), ES cell targeting, TALEN and other technologies, has the characteristics of high efficiency, high speed, simplicity and economy, has very wide application prospects in the aspects of model construction and the like, and the CRISPR/Cas-mediated genome editing technology becomes a research hotspot due to the characteristics of simple operation, high specificity and capability of editing a plurality of genes simultaneously. For mycobacteria, the traditional homologous recombination technology still has the defects of low efficiency, complex steps and the like, so the CRISPR/Cas technology is constructed in wild strain mycobacteria.
Disclosure of Invention
In order to solve the problems, the invention provides a construction method of a Mycobacterium genetic engineering bacterium, which is characterized in that a stable and efficient CRISPR/Cas9 gene editing system is constructed in a wild strain Mycobacterium Mycobacterium sp.LY-1 which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.13031 for the first time, and the strain is applied to the metabolic modification of the bacterium to obtain a plurality of genetic engineering strains, so that the economic benefit of huge steroid medicines can be obtained.
The invention aims to provide a construction method of a mycobacterium genetic engineering bacterium, which comprises the following steps:
(1) synthesizing a codon-optimized Cas protein expression frame for mycobacteria as shown in SEQ ID NO.1 and a sgRNAscaffold expression frame as shown in SEQ ID NO.2, and designing and synthesizing a sgRNA fragment by taking a target gene as a target point;
(2) cloning the Cas9 protein expression cassette in the step (1) into a starting vector;
(3) extracting mycobacteria genome DNA, and amplifying by using DNA polymerase and primers to obtain upstream and downstream gene segments of a target gene as a gene editing and repairing sequence;
(4) connecting a sgRNA fragment designed and synthesized by taking a target gene as a target site to the vector obtained in the step (2), and then connecting the gene editing and repairing sequence prepared in the step (3) to obtain the mycobacteria genome editing vector;
(5) and (5) transforming the gene editing vector obtained in the step (4) into mycobacteria, and culturing and screening to obtain the mycobacteria genetic engineering bacteria.
Further, in the step (2), the starting vector is pMV 261.
Further, in step (2), the Cas9 protein expression cassette and the starting vector pMV261 are linked by a nucleotide sequence shown in SEQ id No. 16.
The second purpose of the invention is to provide the mycobacterium genetic engineering bacteria constructed by the construction method.
Further, the genetically engineered bacterium is knocked out of 3-sterone-delta1Genes encoding dehydrogenase and 17 β -hydroxysteroid dehydrogenase, and overexpressing 3-sterone-9 α -hydroxylase.
Further, the 3-sterone-delta1The nucleotide sequence of the coding gene of the dehydrogenase is shown in SEQ-ID-NO. 8.
Further, the nucleotide sequence of the coding gene of the 17 β -hydroxysteroid dehydrogenase is shown as SEQ ID NO. 9.
Furthermore, the nucleotide sequence of the coding gene of the 3-sterone-9 α -hydroxylase is shown in SEQ ID NO. 15.
Furthermore, the host bacterium of the mycobacterium genetic engineering bacterium is mycobacterium Mycobacterium sp.LY-1.
The third purpose of the invention is to provide the application of the mycobacterium genetic engineering bacteria in the production of 9 α -OH-AD.
The invention has the beneficial effects that:
in the system, after the recombinant plasmid is transformed into a mycobacterium LY-1 cell, the sgRNA can recognize a specific region on a genome and guide Cas9 protein to be combined to a target site, and under the action of the protein, a gap of double-strand break is formed at the target site, and most cells die: the homologous repair sequence introduced with the recombinant plasmid is integrated to the gap through double exchange under the action of recombinase, so that cells successfully repaired by gene homology survive, and artificially designed genotypes such as gene inactivation, foreign gene insertion and the like are formed.
The CRISPR-Cas9 system constructed by the invention has the knockout efficiency of 60% aiming at the same gene, and is more convenient and efficient compared with the existing mycobacteria gene editing mode.
Drawings
FIG. 1 is a schematic diagram of the starting vector pMV 261;
FIG. 2 is a schematic representation of the vector pMVC;
FIG. 3 is a graph showing the results of the molar yield and biomass of 9 α -OH-AD from the engineered bacteria.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1:
a preparation method of a mycobacterium LY-1 genome editing vector based on a CRISPR-Cas9 system;
synthesizing a Cas9 protein expression frame (SEQ ID No.1), a sgRNA expression frame (SEQ ID No.2) and a kstd3-sgRNA fragment (SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7) which are subjected to codon optimization aiming at mycobacteria;
(1) the synthesized Cas protein mutexpression cassette T-A was cloned from pMD19-T-simple vector to obtain plasmid T-Cas 9. Then amplifying the fragment by using a primer P1 (5'-gaaacagaattaattaagcttTGGAAATCTAGAGGTGACCACAAC-3', SEQ ID NO.17) and a primer P2 (5'-caaaacagccaagctgaattcTTAGTCGCCACCCAGCTGG-3', SEQ ID NO.18), and after the gel is recovered, seamlessly splicing the gel to a pMV261 digested by BamH I and EcoR I through 5'-cggaggaatcacttcgca-3' (SEQ ID NO.16) to obtain a plasmid pMV261-Cas 9;
(2) amplifying sgRNA designed with kstd3 as a target by using a Primer P3 (5'-CCCAAGCTTTCGCGGTGAAAGACATGTTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC-3', SEQ ID NO.19) and a Primer P4 (5'-CCCAAGCTTAAAAAACAAGTTGATAACGGACTAGCCTTATTT-3', SEQ ID NO.20), and amplifying by using a Primer star DNA polymerase to obtain a sgRNA scaffold expression cassette with a target N20;
reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min and then entering the next cycle: denaturation at 95 ℃ for 30s, annealing at 68 ℃ for 30s, and extension at 72 ℃ for 15s, for 34 cycles; extending for 5min at 72 ℃, and keeping the temperature at 12 ℃;
purifying the amplified product, carrying out HindIII enzyme digestion, and connecting the product into a HindIII site of a plasmid pMV261-Cas 9;
(3) extracting genomic DNA of mycobacterium LY-1, and respectively amplifying 500bp upstream and downstream of kstd3 by using Primer Star DNA polymerase and Primer P5 (5'-tggccgcggtgatcagctagcGGCTGGTACTGGACGGGTG-3', SEQ ID NO.21), Primer P6 (5'-aagcgctcatTAGGTAACGCTGCCTCCCG-3', SEQ ID NO.22), Primer P7 (5'-gcgttacctaATGAGCGCTTGCGCGAAG-3', SEQ ID NO.23) and Primer P8 (5'-aacgtcgctttgttggctagcAGGATCGAATTCCATTGCCA-3', SEQ ID NO.24) to serve as homologous repair sequences;
the 3-sterone-delta1The gene sequence of the dehydrogenase is shown as SEQ-ID-NO. 8.
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min and then entering the next cycle: denaturation at 95 ℃ for 30s, annealing at 55.9 ℃/56.6 ℃ for 30s, extension at 72 ℃ for 30s, and 34 cycles; extending for 5min at 72 ℃, and keeping the temperature at 12 ℃.
The amplification product was purified and ligated into the NheI site of sgRNA-carrying plasmid pMV261-Cas9 prepared above. Obtaining the mycobacterium LY-1 genome editing vector pMVC 1;
a mycobacterium LY-1 genome editing system based on a CRISPR-Cas9 system containing the mycobacterium LY-1 genome editing vector based on the CRISPR-Cas9 system, and the preparation method of the mycobacterium LY-1 genome editing system comprises the following steps: electrically transforming a mycobacterium LY-1 genome editing vector pMVC1 based on a CRISPR-Cas9 system into an electrically transformed sensing mycobacterium LY-1 cell, and then culturing to obtain the mycobacterium LY-1 genome editing system;
(1) inoculating plasmid DNA into the prepared mycobacteria LY-1 competent cells; placing on ice for 30min, transferring cells into a 2mm electric transformation cup, continuously shocking for 2 times by an eppendorf electric transformation instrument at 2.5kV, rapidly adding 800 μ l of fresh mycobacteria seed solution after shocking, restoring and culturing at 30 ℃ and 120rpm for 3-4h, coating a kanamycin-resistant plate containing 10 μ g/ml, and verifying grown transformants after culturing at 30 ℃ for 5-7 d;
(2) carrying out colony PCR detection on the obtained recombinant mycobacterium LY-1 positive transformant carrying the pMVC1 plasmid by using a specific primer P9 (5'-AAGCGAAGGCGGAGGGGCCGTGGTGCT-3', SEQ ID NO.25) and a primer P10 (5'-CGCGAGGTCGTTGTGCAACCAGTTGATC-3', SEQ ID NO.26) aiming at the upstream/downstream region of a homologous arm on a genome, detecting the transformant with the changed size of a kstd3 fragment by PCR, recovering an amplified fragment of the transformant, sending the amplified fragment to Nanjing Jinweizhi Biotech company for sequencing, comparing a sequencing result with an original gene, and detecting whether the kstd3 gene is successfully knocked out;
(3) picking the single colony with successful knockout into a non-resistant seed culture medium for culture and passage to eliminate the pMVC1 plasmid and obtain the non-3-sterone-delta1Dehydrogenase-active strains, deposited and used for the next round of gene editing.
The successful-edited mycobacterium LY-1 is subjected to a sterol conversion experiment under the culture conditions of 30 ℃, 120rpm and 168 hours, the mixture is extracted by ethyl acetate, dried in vacuum and redissolved, and the result of the sterol conversion reaction is determined by HPLC, so that the molar yield of 9 α -OH-AD reaches 47 percent and is 12 percent higher than that of the wild mycobacterium LY-1, as shown in figure 3 and experimental group 1.
Example 2:
by utilizing the constructed gene editing system based on CRISPR/Cas9, on the basis of knocking out a gene kstd3, 17 β -hydroxysteroid dehydrogenase (17 β -hsd) is knocked out and edited simultaneously, and a gene of 3-sterone-9 α -hydroxylase (kshA) is inserted and edited;
the gene sequence of the 17 β -hydroxysteroid dehydrogenase is shown in SEQ-ID-NO. 9.
Synthesizing a Cas9 protein expression frame (SEQ ID No.1), an sgRNA expression frame (SEQ ID No.2) and a 17 β -hsd-sgRNA fragment (SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13 and SEQ ID No.14) which are subjected to codon optimization aiming at mycobacteria;
(1) the synthesized Cas protein mutexpression cassette T-A was cloned from pMD19-T-simple vector to obtain plasmid T-Cas 9. Amplifying the fragment by using a primer P1 (5'-gaaacagaattaattaagcttTGGAAATCTAGAGGTGACCACAAC-3') and a primer P2 (5'-caaaacagccaagctgaattcTTAGTCGCCACCCAGCTGG-3'), and seamlessly splicing the recovered gel to the pMV261 digested by BamH I and EcoR I to obtain a plasmid pMVC261-Cas 9;
(2) amplifying sgRNA designed by taking 17 β -hsd as a target by using a Primer P11 (5'-CCCAAGCTTCTTGGGGGCGAACCGCTGACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC-3', SEQ ID NO.27) and a Primer P12 (5'-CCGACGCGTAAAAAACAAGTTGATAACGGACTAGCCTTATTT-3', SEQ ID NO.28), and amplifying by using a Primer star DNA polymerase to obtain a sgRNA scaffold expression cassette with a target N20;
reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min and then entering the next cycle: denaturation at 95 ℃ for 30s, annealing at 68 ℃ for 30s, and extension at 72 ℃ for 15s, for 34 cycles; extending for 5min at 72 ℃, and keeping the temperature at 12 ℃;
(3) purifying the amplified product, carrying out HindIII enzyme digestion, and connecting the product into a HindIII site of a plasmid pMV261-Cas 9;
extracting genomic DNA of mycobacterium LY-1, and amplifying 500bp and kshA respectively on the upstream and downstream of 17 β -hsd as homologous repair sequences by using Primer Star DNA polymerase, Primer P13 (5'-tggccgcggtgatcagctagcGCGCATCGGTGAGCAGCT-3', SEQ ID NO.29), Primer P14 (5'-ggtagtcacTCTAGCTTCCTTTCCAAAAATGACC-3', SEQ ID NO.30), Primer P15 (5'-gtcaagctgaGAGTGAGTTCCGGCGTGATC-3', SEQ ID NO.31), Primer P16 (5'-aacgtcgctttgttggctagcCGCCATCTCGAGGATGCG-3', SEQ ID NO.32), Primer P17 (5'-aggaagctagaGTGACTACCGAACATGCCGG-3', SEQ ID NO.33) and Primer P18 (5'-gaactcactcTCAGCTTGACTGCGCGGT-3', SEQ ID NO. 34);
the gene sequence of the 3-sterone-9 α -hydroxylase is shown in SEQ-ID-NO. 15.
The reaction conditions are respectively as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 57.7 ℃ for 30s, extension at 72 ℃ for 15s, and 34 cycles; extending for 5min at 72 ℃, and keeping the temperature at 12 ℃; pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 55.8 ℃ for 30s, extension at 72 ℃ for 1min, and 34 cycles; extending for 5min at 72 ℃, and keeping the temperature at 12 ℃; pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 56.5 ℃ for 30s, extension at 72 ℃ for 15s, and 34 cycles; extending for 5min at 72 ℃, and keeping the temperature at 12 ℃;
(4) and (4) purifying and connecting the amplification product obtained in the step (3) into the Nhe I site of the plasmid pMV261-Cas9 with the sgRNA prepared in the step (3). Obtaining the mycobacterium LY-1 genome editing vector pMVC 2;
a mycobacterium LY-1 genome editing system based on a CRISPR-Cas9 system containing the mycobacterium LY-1 genome editing vector based on the CRISPR-Cas9 system, and the preparation method of the mycobacterium LY-1 genome editing system comprises the following steps: electrically transforming a mycobacterium LY-1 genome editing vector pMVC2 based on a CRISPR-Cas9 system into an electrically transformed sensing mycobacterium LY-1 cell, and then culturing to obtain the mycobacterium LY-1 genome editing system;
(1) inoculating plasmid DNA into the prepared mycobacteria LY-1 competent cells; placing on ice for 30min, transferring cells into a 2mm electric transformation cup, continuously shocking for 2 times by an eppendorf electric transformation instrument at 2.5kV, rapidly adding 800 μ l of fresh mycobacteria seed solution after shocking, restoring and culturing at 30 ℃ and 120rpm for 3-4h, coating a kanamycin-resistant plate containing 10 μ g/ml, and verifying grown transformants after culturing at 30 ℃ for 5-7 d;
(2) carrying out colony PCR detection on the obtained recombinant mycobacterium LY-1 positive transformant carrying the pMVC2 plasmid by using a specific primer P19 (5'-GCGCATCGGTGAGCAGCTGAGTC-3', SEQ ID NO.35) and a primer P20 (5'-CGCCATCTCGAGGATGCGC-3', SEQ ID NO.36) aiming at the upstream/downstream region of a homologous arm on a genome, detecting the transformant with the position size of a 17 β -hsd fragment changed by PCR, recovering an amplified fragment of the transformant, sending the amplified fragment to Nanjing Jinweizhi Biotech company for sequencing, comparing a sequencing result with an original gene, and detecting whether the 17 β -hsd gene is successfully edited;
(3) and picking the single colony with successful knockout to a non-resistant seed culture medium for culture and passage to eliminate the pMVC2 plasmid, obtaining a strain without the activity of 3-sterone-delta 1-dehydrogenase and 17 β -hydroxysteroid dehydrogenase and with high activity of 3-sterone-9 α -hydroxylase, and preserving and using the strain for next round of gene editing.
The successful-edited mycobacterium LY-1 is subjected to a sterol conversion experiment under the culture conditions of 30 ℃, 120rpm and 168 hours, the mixture is extracted by ethyl acetate, dried in vacuum and redissolved, and the result of the sterol conversion reaction is determined by HPLC, so that the molar yield of 9 α -OH-AD reaches 65 percent and is improved by 55 percent compared with that of the wild mycobacterium LY-1, as shown in the attached figure 3 and an experimental group 5.
Example 3:
3-sterone-delta simultaneously by utilizing a CRISPR/Cas 9-based gene editing system1Knock-out edits were performed with dehydrogenase (kstd3) and 17 β -hydroxysteroid dehydrogenase (17 β -hsd).
The 3-sterone-delta1The gene sequence of the dehydrogenase is shown as SEQ-ID-NO. 8.
The gene sequence of the 17 β -hydroxysteroid dehydrogenase is shown in SEQ-ID-NO. 9.
Specific experimental method referring to example 2, the successful edited mycobacterium LY-1 was verified to perform sterol conversion experiment under the culture conditions of 30 ℃, 120rpm, 168h, extracted with ethyl acetate, vacuum dried and redissolved, and the result of sterol conversion reaction was confirmed by HPLC, the molar yield of 9 α -OH-AD reached 47%, which was 12% higher than that of wild mycobacterium LY-1, as shown in fig. 3 and experimental group 2.
Example 4:
3-sterone-delta simultaneously by utilizing a CRISPR/Cas 9-based gene editing system1-a knock-out editing of dehydrogenase (kstd3) and an insertion editing of the gene for 3-sterone-9 α -hydroxylase (kshA).
The gene sequence of the 3-ketosteroid-delta 1-dehydrogenase is shown in SEQ-ID-NO. 8.
The gene sequence of the 3-sterone-9 α -hydroxylase is shown in SEQ-ID-NO. 15.
Specific experimental method referring to example 2, the successful edited mycobacterium LY-1 was verified to perform sterol conversion experiment under the culture conditions of 30 ℃, 120rpm, 168h, extracted with ethyl acetate, vacuum dried and redissolved, and the result of sterol conversion reaction was confirmed by HPLC, the molar yield of 9 α -OH-AD reached 58%, which was 38% higher than that of wild mycobacterium LY-1, as shown in fig. 3 and experimental group 3.
Example 5:
the constructed CRISPR/Cas 9-based gene editing system is used for simultaneously carrying out knockout editing on 17 β -hydroxysteroid dehydrogenase (17 β -hsd) and carrying out insertion editing on a gene of 3-sterone-9 α -hydroxylase (kshA).
The gene sequence of the 17 β -hydroxysteroid dehydrogenase is shown in SEQ-ID-NO. 9.
The gene sequence of the 3-sterone-9 α -hydroxylase is shown in SEQ-ID-NO. 15.
Specific experimental method referring to example 2, the successful edited mycobacterium LY-1 was verified to perform sterol conversion experiment under the culture conditions of 30 ℃, 120rpm, 168h, extracted with ethyl acetate, vacuum dried and redissolved, and the result of sterol conversion reaction was confirmed by HPLC, the molar yield of 9 α -OH-AD reached 56%, which is 33% higher than that of wild mycobacterium LY-1, as shown in fig. 3 and experimental group 4.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> university of south of the Yangtze river
<120> construction method and application of mycobacterium genetic engineering bacteria
<160>36
<170>PatentIn version 3.3
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ctggaggagt cgttcctggt ggaggaggat aagaagcacg agcgccaccc gatcttcggt 360
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cgtctggaga acctgatcgc ccagttgccg ggtgaaaaga agaacggcct gttcggcaac 720
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gacgccaagt tgcagctgtc gaaggacacc tacgacgacg acctggacaa cctgctggcc 840
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cagcagctgc cggagaagta taaggagatt tttttcgacc agtcgaagaa cggctacgcg 1080
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gtcaagcagc tgaaggagga ctacttcaag aagatcgagt gcttcgactc ggtggagatc 1740
agcggtgtcg aggatcgttt caacgcgagc ctgggtacgt accacgactt gttgaagatc 1800
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gacttcctga agtcggacgg cttcgccaac cgtaacttca tgcagctgat ccacgacgac 2100
tcgctgacct tcaaggagga catccagaag gcccaggtgt cgggtcaggg tgactcgctg 2160
cacgagcaca tcgccaatct ggccggttcg ccggccatca agaagggtat cctgcaaacc 2220
gtcaaggtcg tcgatgagct ggtcaaggtg atgggccgtc acaagccgga aaacatcgtg 2280
atcgagatgg cgcgcgagaa ccagaccacc cagaagggtc agaagaactc gcgcgagcgt 2340
atgaagcgca tcgaggaagg tatcaaggag ctgggctcgc agatcctgaa ggagcatccg 2400
gtggagaaca cccagctgca aaacgagaag ctgtacctgt actacctgca aaacggccgc 2460
gacatgtacg tggaccagga attggacatc aaccgcctgt cggactacga cgtggaccat 2520
atcgtcccgc agtcgttcat caaggacgac tcgatcgaca acaaggtgct gacccgctcg 2580
gacaagaacc gtggtaagtc ggacaacgtc ccgtcggaag aggtggtgaa gaagatgaag 2640
aactactggc gccagctgct gaacgccaag ttgatcaccc agcgcaagtt cgacaacctg 2700
accaaggccg agcgcggtgg tctgtcggag ctggacaagg ccggtttcat caagcgccag 2760
ctggtcgaaa cccgccagat caccaagcac gtcgcccaga tcctggactc gcgtatgaac 2820
accaagtacg acgagaacga caagctgatc cgcgaggtca aggtcatcac cctgaagtcg 2880
aagctggtca gcgattttcg caaggacttc cagttctaca aggtgcgcga gatcaacaac 2940
taccaccacg cgcacgacgc ctacttgaac gccgtggtgg gtacggcctt gatcaagaag 3000
tacccgaagc tggagtcgga gttcgtgtac ggtgactaca aggtgtacga cgtccgcaag 3060
atgctggcca agtcggaaca ggagatcggc aaggccaccg ccaagtattt cttctactcg 3120
aacatcatga acttcttcaa gaccgagatc accctggcca acggcgaaat ccgtaagcgt 3180
ccgttgatcg agacgaacgg cgagacgggt gagatcgtgt gggataaggg ccgtgacttc 3240
gccactgtgc gtaaggtgct gtcgatgccg caggtgaaca tcgtcaagaa gaccgaggtc 3300
cagaccggcg gtttctcgaa ggaatcgatc ctgccgaagc gcaactcgga caagctgatc 3360
gcccgtaaga aggactggga cccgaagaag tacggcggtt tcgactcgcc gaccgtggcc 3420
tactcggtgc tggtggtggc caaggtggaa aagggtaagt cgaagaagct gaagtcggtc 3480
aaggagctgc tgggcatcac catcatggag cgttcgtcgt tcgagaagaa cccgatcgac 3540
ttcctggagg ccaagggtta caaggaggtg cgtaaggacc tgatcatcaa gctgccgaag 3600
tactcgctgt tcgagctgga gaacggccgt aagcgtatgc tggcctcggc cggtgagctg 3660
caaaagggta atgagctggc gctgccgtcg aagtacgtca atttcctgta cctggcctcg 3720
cactacgaga agctgaaggg ttcgccggag gacaacgagc agaagcagtt gttcgtcgag 3780
cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc gaagcgcgtg 3840
atcctggccg atgccaatct ggataaggtg ctgagcgcgt acaacaagca ccgcgataag 3900
ccgatccgcg agcaggccga gaatatcatc cacctgttca ccctgaccaa cctgggcgcc 3960
ccgaccgcct tcaagtactt cgacaccacc atcgaccgca agcgctacac ctcgaccaag 4020
gaagtcctgg acgccacctt catccaccag tcgatcaccg gactgtacga gacgcgcatc 4080
gacttgtcgc agctgggtgg tgat 4104
<210>2
<211>118
<212>DNA
<213> (Artificial sequence)
<220>
<221>misc_feature
<222>(9)..(28)
<223>n is a, c, g, or t
<400>2
cggaattcnn nnnnnnnnnn nnnnnnnngt tttagagcta gaaatagcaa gttaaaataa 60
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ggatcccg 118
<210>3
<211>20
<212>DNA
<213> (Artificial sequence)
<400>3
<210>4
<211>20
<212>DNA
<213> (Artificial sequence)
<400>4
<210>5
<211>20
<212>DNA
<213> (Artificial sequence)
<400>5
<210>6
<211>20
<212>DNA
<213> (Artificial sequence)
<400>6
<210>7
<211>20
<212>DNA
<213> (Artificial sequence)
<400>7
<210>8
<211>1545
<212>DNA
<213> (Artificial sequence)
<400>8
atgaagtggg ccgacgaatg tgatgtcctg gtcgcggggt ccggcggtgg tggcgtcacc 60
ggcgcataca ccgccgcccg cgaagggctg tcggtgatcc tggtcgaggc cagcgacaaa 120
ttcgggggca ccacagcgta ttccggtggt ggcggggtgt ggttcccgtg caacccggtg 180
ctcacccgcg ccggcaccga cgacaccatc gaggatgcgc tggagtacta ccacgccgtc 240
gtcggggacc ggacccctcg ggagctgcag gagacctacg tccgtggcgg cgccgggctg 300
atcgagtacc tggaagctga cgcctttctg aagttcgccc cgatgccgtg gcccgactac 360
ttcggcaagg cgcccaaggc ccgcaccgac ggtcaacgtc atatcgcggc gcgtccgctc 420
agggtcgaga aggccccgca cctgcgggag ttggtacgcg gcccgctcga cgccgaccgg 480
ctcggcgccg aacagcccga cgactatttc atcggcggcc gggcgttgat cgcccggttc 540
ctcaaagcca tcgagcagta ccagaatgcc tcgcttcggc tcaacacccc gttggtcgag 600
ttggtcgtcg aggacggcat cgtgacgggc gcgatcgtcg aaaccgacgg tgagcgcaag 660
gccatccggg cccggcgcgg tgtgctgctc gccgcgggcg gcttcgaggg caacgacgac 720
ctgcgccgcg aatacggtgt gccgggcgtc gcacgcgaca ccatggggcc gccggccaac 780
ctcgggcatg cgcaccaggc agcgatcgcc gtcggcgccg acgtcgacct gatggagcag 840
gcctggtggt cacctggaat gacccacccc gacgggcgct cggcgttcgc gctgtggttc 900
accggcggca tcttcgtcga ccagaacgga cggcgtttcg tcaacgagtc ggcggcttat 960
gaccggatcg gccgtgcgat cctgccgcgg ttggccgacg ggtcgatgac gttgccgtac 1020
tggatgatct acgacgaccg ggagggggaa atccctcccg tcaaggccac caacgtatcg 1080
atggtcgaca cacagcaata cgtcgacgcc ggcttgtggc acacggccga caccctcgaa 1140
gagctggccg cgaagatcgg tgtccccgcc gagaacctgg tcgccaccgt ggagcgtttc 1200
aacgaattcg tcgtcgccgg caccgacgag gatttcgggc gtggcgacga ggcctacgac 1260
cgggccttct cgggcggtgc gtccccgctc gtcgcgatcg agaagggccc tttccacgcc 1320
gctgcgttcg gcatttccga tctgggcaca aaggggggat tgcgtaccga caccgcagcc 1380
cgggtactcg acaccgacgg tcaggtgatc gcaggactgt acgcggcggg aaacacgatg 1440
gccgccccca gcggcaccgc ctacccgggc ggcggaaatc cgatcggaac cagcatgctg 1500
ttcagccacc tcgcggtgaa agacatgtta gggatggact tgtga 1545
<210>9
<211>767
<212>DNA
<213> (Artificial sequence)
<400>9
atggagatcg aaggcaagaa ggcgatcgtc gtcggcggcg cgtccggctt tggccgcgcg 60
accgccgagg cgttggccaa gcggggcgcc agcgtggctg tgctggaccg gccgcaatcc 120
aagggccagg aagtggccga cgcgatcggc ggctcgttct tcgccgtcga cgtcacggac 180
ttcgacggta ccgaaaaggt gctggaagag gccgtcgcgg cgctgggtgg tctgcacatc 240
atcgtgacca ccgcaggtgg cggcatcggc gagcgcacca tcaagaagga cggcccgcac 300
agcctggatt cgttccgttc caccatcgac ctcaacctca tcggcacgtt caacatcagc 360
cggctggcgg cgtggcacat gagcaagaac gagccggtcg acgccgaggc cgaggagcgc 420
ggcgtcatca tcaacaccgc ctcgatcgcc gcgttcgagg gccagatcgg tcaggtcgcc 480
tacaccgcgt ccaaggccgc gatcgccggc atgtgcctga ccatggcgcg cgacctggga 540
gtctgggaat ccgcgtgctg gccatcgcgc cgagcctgtt cgccaccggt ctgaccgagg 600
gcattcccga cgagttcgcc gcggtgctga ccaaggacgc cgcgttcccc aagcgtctgg 660
gcaagcccga ggagtacgcc aagctcgccg tggcgatcgc cgagaacgcg atgctcaacg 720
gccagtgtct gcgtttggac gccggtcagc ggttcgcccc caagtag 767
<210>10
<211>20
<212>DNA
<213> (Artificial sequence)
<400>10
<210>11
<211>20
<212>DNA
<213> (Artificial sequence)
<400>11
<210>12
<211>20
<212>DNA
<213> (Artificial sequence)
<400>12
<210>13
<211>20
<212>DNA
<213> (Artificial sequence)
<400>13
<210>14
<211>20
<212>DNA
<213> (Artificial sequence)
<400>14
<210>15
<211>1164
<212>DNA
<213> (Artificial sequence)
<400>15
gtgactaccg aacatgccgg catcagagag atcgacaccg gcgccctgcc agaccgctat 60
gcgcggggtt ggcactgcct gggaccggtc aagaacttcc ttgacggtca gccgcactct 120
gtcgagatct tcggcaccaa gttggtcgtg ttcgccgaca ccaagggcga gttgaagatt 180
ctcgacggct actgccggca catgggcggt gacctgtcgc agggcaccat caagggtgac 240
gaggtggcct gcccgttcca cgactggcgc tggggcggcg acggcaagtg caagctggtg 300
ccctacgcca agcgcacccc acgcctggcc cgcacccggg cctggcacac cgatgtccgc 360
ggtggcctgc tgttcgtatg gcacgaccac gagggcaacg acccgcagcc cgaggtccgg 420
attcccgaga tccccgaggc cgccagcgac gagtggaccg agtggcagtg gaactcgatg 480
ctgatcgagg gcagcaactg ccgcgagatc atcgacaacg tcaccgacat ggcgcacttc 540
ttctacatcc acttcggtct gccgacgtac ttcaagaacg tcttcgaggg gcacatcgcc 600
tcgcagtacc tgcacaacgt cgggcgtcag gacatcggcg gcatggggac gcagtacggc 660
gagtcgcacc tggactccga agcctcctac ttcggcccgt ccttcatgat caactggctg 720
cacaacaact acagcggcta caaggcagag tcgatcctga tcaactgcca ctacccggtg 780
acgcaggact cgttcatgct gcagtggggc gtcatcgtcg aaaagcccaa gggtatggac 840
gagaagacca cgcagaagct ggccaacgcg atgaccgacg gcgtcagcca gggcttcctg 900
caggacgtgg agatctggaa gcacaagacc cgcatcgaca acccgctgct ggtcgaggag 960
gacggcgccg tctaccagat gcgccgctgg taccagcagt tctacgtcga cgtcgccgac 1020
atcacgccgg acatgaccga ccggttcgag atggagatcg acaccaccgc ggccaacgag 1080
aagtggcacg tggaggtcga ggagaacctg aagatccagg ccgagcagaa agcggccgag 1140
aaagaaaccg cgcagtcaag ctga 1164
<210>16
<211>18
<212>DNA
<213> (Artificial sequence)
<400>16
cggaggaatc acttcgca 18
<210>17
<211>45
<212>DNA
<213> (Artificial sequence)
<400>17
gaaacagaat taattaagct ttggaaatct agaggtgacc acaac 45
<210>18
<211>40
<212>DNA
<213> (Artificial sequence)
<400>18
caaaacagcc aagctgaatt cttagtcgcc acccagctgg 40
<210>19
<211>70
<212>DNA
<213> (Artificial sequence)
<400>19
cccaagcttt cgcggtgaaa gacatgttag ttttagagct agaaatagca agttaaaata 60
<210>20
<211>42
<212>DNA
<213> (Artificial sequence)
<400>20
cccaagctta aaaaacaagt tgataacgga ctagccttat tt 42
<210>21
<211>40
<212>DNA
<213> (Artificial sequence)
<400>21
tggccgcggt gatcagctag cggctggtac tggacgggtg 40
<210>22
<211>29
<212>DNA
<213> (Artificial sequence)
<400>22
aagcgctcat taggtaacgc tgcctcccg 29
<210>23
<211>28
<212>DNA
<213> (Artificial sequence)
<400>23
gcgttaccta atgagcgctt gcgcgaag 28
<210>24
<211>41
<212>DNA
<213> (Artificial sequence)
<400>24
aacgtcgctt tgttggctag caggatcgaa ttccattgcc a 41
<210>25
<211>27
<212>DNA
<213> (Artificial sequence)
<400>25
aagcgaaggc ggaggggccg tggtgct 27
<210>26
<211>28
<212>DNA
<213> (Artificial sequence)
<400>26
cgcgaggtcg ttgtgcaacc agttgatc 28
<210>27
<211>70
<212>DNA
<213> (Artificial sequence)
<400>27
cccaagcttc ttgggggcga accgctgacg ttttagagct agaaatagca agttaaaata 60
<210>28
<211>42
<212>DNA
<213> (Artificial sequence)
<400>28
ccgacgcgta aaaaacaagt tgataacgga ctagccttat tt 42
<210>29
<211>39
<212>DNA
<213> (Artificial sequence)
<400>29
tggccgcggt gatcagctag cgcgcatcgg tgagcagct 39
<210>30
<211>34
<212>DNA
<213> (Artificial sequence)
<400>30
ggtagtcact ctagcttcct ttccaaaaat gacc 34
<210>31
<211>30
<212>DNA
<213> (Artificial sequence)
<400>31
gtcaagctga gagtgagttc cggcgtgatc 30
<210>32
<211>39
<212>DNA
<213> (Artificial sequence)
<400>32
aacgtcgctt tgttggctag ccgccatctc gaggatgcg 39
<210>33
<211>31
<212>DNA
<213> (Artificial sequence)
<400>33
aggaagctag agtgactacc gaacatgccg g 31
<210>34
<211>28
<212>DNA
<213> (Artificial sequence)
<400>34
gaactcactc tcagcttgac tgcgcggt 28
<210>35
<211>23
<212>DNA
<213> (Artificial sequence)
<400>35
gcgcatcggt gagcagctga gtc 23
<210>36
<211>19
<212>DNA
<213> (Artificial sequence)
<400>36
cgccatctcg aggatgcgc 19
Claims (10)
1. A construction method of a mycobacterium genetic engineering bacterium is characterized by comprising the following steps:
(1) synthesizing a Cas protein expression frame with a nucleotide sequence shown as SEQ ID NO.1 and optimized by a codon aiming at mycobacteria and a sgRNA scaffold expression frame with a nucleotide sequence shown as SEQ ID NO.2, and designing and synthesizing a sgRNA fragment by taking a target gene as a target point;
(2) cloning the Cas9 protein expression cassette in the step (1) into a starting vector;
(3) extracting mycobacteria genome DNA, and amplifying by using DNA polymerase and primers to obtain upstream and downstream gene segments of a target gene as a gene editing and repairing sequence;
(4) connecting a sgRNA fragment designed and synthesized by taking a target gene as a target site to the vector obtained in the step (2), and then connecting the gene editing and repairing sequence prepared in the step (3) to obtain the mycobacteria genome editing vector;
(5) and (4) transforming the gene editing vector obtained in the step (4) into mycobacteria, and culturing and screening to obtain the mycobacterium genetic engineering bacteria.
2. The method according to claim 1, wherein in step (2), the starting vector is pMV 261.
3. The construction method according to claim 2, characterized in that in step (2), the Cas9 protein expression cassette is linked with the starting vector pMV261 by a nucleotide sequence shown as SEQ ID No. 16.
4. A genetically engineered Mycobacterium produced by the method of claim 1.
5. The genetically engineered mycobacterium of claim 4, wherein the genetically engineered mycobacterium has 3-sterone- △ knocked out1Genes encoding dehydrogenase and 17 β -hydroxysteroid dehydrogenase, and overexpressing 3-sterone-9 α -hydroxylase.
6. The genetically engineered Mycobacterium of claim 5, wherein said 3-sterone- △1The nucleotide sequence of the coding gene of the dehydrogenase is shown in SEQ-ID-NO. 8.
7. The genetically engineered mycobacterium of claim 5, wherein the nucleotide sequence of the gene encoding 17 β -hydroxysteroid dehydrogenase is shown in SEQ ID No. 9.
8. The genetically engineered mycobacterium of claim 5, wherein the nucleotide sequence of the gene encoding 3-sterone-9 α -hydroxylase is shown in SEQ ID No. 15.
9. The genetically engineered Mycobacterium of claim 5, wherein the host bacterium is Mycobacterium mycobacter sp.ly-1.
10. Use of a genetically engineered mycobacterium of any one of claims 4-9 to produce 9 α -hydroxyandrost-4-ene-3, 17-dione (9 α -OH-AD).
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Cited By (6)
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|---|---|---|---|---|
| CN111454871A (en) * | 2020-03-03 | 2020-07-28 | 天津大学 | Recombinant mycobacterium with high androstenedione yield, construction method and application |
| CN111484964A (en) * | 2020-03-14 | 2020-08-04 | 天津大学青岛海洋技术研究院 | High-yield 9-OHAD mycobacterium and construction method thereof |
| CN112592904A (en) * | 2020-12-31 | 2021-04-02 | 江南大学 | 17 beta-hydroxysteroid dehydrogenase mutant of mycobacterium and heterologous expression thereof |
| CN112813041A (en) * | 2020-12-31 | 2021-05-18 | 江南大学 | 17 beta-hydroxysteroid dehydrogenase mutant of mycobacterium, engineering bacterium and application of mutant and engineering bacterium |
| CN114085859A (en) * | 2021-11-10 | 2022-02-25 | 浙江工业大学 | Gene editing method and system for new mycobacterium aurum engineering bacteria |
| WO2022126219A3 (en) * | 2020-12-18 | 2022-09-29 | Instituto Butantan | Process for construction of a single vector comprising cas9 and sgrna for mycobacterial genome modifications |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111454871A (en) * | 2020-03-03 | 2020-07-28 | 天津大学 | Recombinant mycobacterium with high androstenedione yield, construction method and application |
| CN111484964A (en) * | 2020-03-14 | 2020-08-04 | 天津大学青岛海洋技术研究院 | High-yield 9-OHAD mycobacterium and construction method thereof |
| WO2022126219A3 (en) * | 2020-12-18 | 2022-09-29 | Instituto Butantan | Process for construction of a single vector comprising cas9 and sgrna for mycobacterial genome modifications |
| CN112592904A (en) * | 2020-12-31 | 2021-04-02 | 江南大学 | 17 beta-hydroxysteroid dehydrogenase mutant of mycobacterium and heterologous expression thereof |
| CN112813041A (en) * | 2020-12-31 | 2021-05-18 | 江南大学 | 17 beta-hydroxysteroid dehydrogenase mutant of mycobacterium, engineering bacterium and application of mutant and engineering bacterium |
| CN112592904B (en) * | 2020-12-31 | 2023-02-07 | 江南大学 | 17 beta-hydroxysteroid dehydrogenase mutant of mycobacterium and heterologous expression thereof |
| CN114085859A (en) * | 2021-11-10 | 2022-02-25 | 浙江工业大学 | Gene editing method and system for new mycobacterium aurum engineering bacteria |
| CN114085859B (en) * | 2021-11-10 | 2024-02-13 | 浙江工业大学 | Gene editing method and system for new mycobacterium aurum engineering bacteria |
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|---|---|
| CN110791468B (en) | 2021-11-23 |
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