CN117778280A - Chassis strain suitable for heterologous biosynthesis of antibacterial material myxin, construction method and application thereof - Google Patents
Chassis strain suitable for heterologous biosynthesis of antibacterial material myxin, construction method and application thereof Download PDFInfo
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- CN117778280A CN117778280A CN202311271128.7A CN202311271128A CN117778280A CN 117778280 A CN117778280 A CN 117778280A CN 202311271128 A CN202311271128 A CN 202311271128A CN 117778280 A CN117778280 A CN 117778280A
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- 241000251748 Myxinidae Species 0.000 title claims abstract description 52
- JIDVGUQUQSOHOL-UHFFFAOYSA-N myxin Chemical compound C1=CC=C2[N+]([O-])=C3C(OC)=CC=CC3=[N+]([O-])C2=C1O JIDVGUQUQSOHOL-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 21
- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 13
- 238000010276 construction Methods 0.000 title abstract description 6
- 239000000463 material Substances 0.000 title description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 239000000126 substance Substances 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 11
- 241000589516 Pseudomonas Species 0.000 claims abstract description 10
- 238000002744 homologous recombination Methods 0.000 claims abstract description 10
- 230000006801 homologous recombination Effects 0.000 claims abstract description 9
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 8
- VEPOHXYIFQMVHW-XOZOLZJESA-N 2,3-dihydroxybutanedioic acid (2S,3S)-3,4-dimethyl-2-phenylmorpholine Chemical compound OC(C(O)C(O)=O)C(O)=O.C[C@H]1[C@@H](OCCN1C)c1ccccc1 VEPOHXYIFQMVHW-XOZOLZJESA-N 0.000 claims abstract description 7
- PCNDJXKNXGMECE-UHFFFAOYSA-N Phenazine Natural products C1=CC=CC2=NC3=CC=CC=C3N=C21 PCNDJXKNXGMECE-UHFFFAOYSA-N 0.000 claims abstract description 7
- MJALVONLCNWZHK-UHFFFAOYSA-N phenazine-1,6-dicarboxylic acid Chemical compound C1=CC=C2N=C3C(C(=O)O)=CC=CC3=NC2=C1C(O)=O MJALVONLCNWZHK-UHFFFAOYSA-N 0.000 claims description 38
- 239000012634 fragment Substances 0.000 claims description 13
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims description 7
- 238000009629 microbiological culture Methods 0.000 claims description 6
- 238000011144 upstream manufacturing Methods 0.000 claims description 6
- 238000012217 deletion Methods 0.000 claims description 4
- 244000005700 microbiome Species 0.000 claims description 4
- 108091008146 restriction endonucleases Proteins 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 3
- 230000021615 conjugation Effects 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 238000012262 fermentative production Methods 0.000 claims 1
- 239000002243 precursor Substances 0.000 abstract description 5
- 238000011161 development Methods 0.000 abstract description 4
- 230000003115 biocidal effect Effects 0.000 abstract description 2
- 241000863031 Lysobacter Species 0.000 abstract 1
- 241001660189 Lysobacter antibioticus Species 0.000 abstract 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 abstract 1
- JGCSKOVQDXEQHI-UHFFFAOYSA-N phenazine-1-carboxylic acid Chemical compound C1=CC=C2N=C3C(C(=O)O)=CC=CC3=NC2=C1 JGCSKOVQDXEQHI-UHFFFAOYSA-N 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 239000000575 pesticide Substances 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
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- 238000003776 cleavage reaction Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 208000035404 Autolysis Diseases 0.000 description 2
- 206010057248 Cell death Diseases 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960002518 gentamicin Drugs 0.000 description 2
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- 239000007788 liquid Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
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- 239000007787 solid Substances 0.000 description 2
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- 239000005720 sucrose Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 241000726119 Acidovorax Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
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- 238000012224 gene deletion Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
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- 238000006467 substitution reaction Methods 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a chassis strain suitable for heterologous biosynthesis of an antibacterial substance myxin, and a construction method and application thereof. Myxin is a phenazine antibacterial substance produced by the antibiotic lysobacter (Lysobacter antibioticus) OH13, but low yields limit the industrial development thereof. The invention uses homologous recombination method to knock out Pseudomonas aeruginosaPseudomonas chlororaphis) Phenazine synthesis related genes on TC3 strain genomephzA2Obtaining strain deltaphzA2The strain successfully synthesizes a synthesis precursor phenazine-1, 6-The dicarboxylic acid PDC provides a suitable chassis strain for subsequent heterologous biosynthesis of myxin in Pseudomonas.
Description
Technical Field
The invention belongs to the field of microbial genetic engineering, and particularly relates to a chassis strain suitable for heterologous biosynthesis of an antibacterial material myxin, and a construction method and application thereof.
Background
In recent years, the mass application of chemical pesticides and the environmental and food safety problems caused by the chemical pesticides have raised more and more social concerns, and also provide great development opportunities for the development of bio-pesticides that are both ecologically safe and environmentally compatible. The biological pesticide industry is increasingly paid attention to as the sun-facing industry of modern agriculture, and becomes one of the hot spots of research in the scientific and technological world and the industry in China. Microbial pesticides and metabolic product pesticides are two important biological pesticides, the former mainly uses living bodies of microorganisms as effective components, and the latter uses active secondary metabolic products of microorganisms as effective components. myxin is a team on Xu Gaoge from Acidovorax avis @Lysobacter antibioticus) The structure of the phenazine oxynitride obtained by separation in OH13 is 1-hydroxy-6-methoxy-5, 10-dioxyphenazine, has stronger antagonistic activity on various plant pathogenic bacteria, especially on Xanthomonas oryzae, and has the potential of being developed into a novel biological bactericide. The American Roche company developed myxin as a copper chelated form as a veterinary antibiotic, but limited the large-scale use of this product due to the low yield of myxin from microbial sources. In wild-type strain OH13, the yield of myxin was extremely low (only 1.5. Mu.g/mL)The requirement of industrial production can not be met far. Thus, increasing yield is a bottleneck in achieving myxin industrialization and large-scale applications.
Since the original production strain OH13 of myxin is easy to cause autolysis with complex mechanism, the myxin cannot be continuously synthesized and accumulated in a large amount, and the way of improving the yield of the myxin by directly carrying out genetic improvement on the OH13 is limited. Chemical synthesis is another way to increase the yield, but the chemical synthesis has higher difficulty, higher cost and easy pollution at present. According to the results of previous studies, the use of synthetic biology techniques to reconstruct the biosynthetic pathway of myxin in chassis strains more suitable for expression of myxin is currently the realistic and most efficient pathway to increase its yield.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a chassis strain suitable for heterologous biosynthesis of an antibacterial substance myxin, a construction method and application thereof, and the obtained chassis strain provides a new path for solving bottleneck problems of myxin industrialization and large-scale application by utilizing a synthetic biological technology. The invention is based on pseudomonas aeruginosaPseudomonas chlororaphis) The strain constructed by TC3 has a certain tolerance to myxin and can generate a synthetic precursor substance of the myxin, namely the phenazine-1, 6-dicarboxylic acid PDC, thereby laying a foundation for the subsequent heterologous biosynthesis of the myxin in the strain, solving the problem of low yield of the myxin caused by autolysis of the strain originally produced by the myxin and laying a foundation for the industrialized development of the myxin.
In order to solve the problems in the prior art, the invention adopts the technical scheme that:
chassis strain suitable for heterologous biosynthesis of antibacterial material myxin, wherein the chassis strain adopts pseudomonas aeruginosaPseudomonas chlororaphis) TC3 as an original strain and knocks out phenazine synthesis genes by a homologous recombination methodphzA2The starting strain Pseudomonas aeruginosaPseudomonas chlororaphis) TC3 was deposited in China general microbiological culture Collection center CGMCC (China general microbiological culture Collection center) at about 05 and 19 months 2022, and the accession number is: CGMCC No.24936, preservation address: beijing city, chaoyang district, northThe national academy of sciences of China, proc.3, chenxi Lu 1.
As an improvement, the code is thatphzA2The nucleotide sequence of the gene is shown as SEQ ID No. 1.
The preparation method of the chassis strain suitable for heterologous biosynthesis of the antibacterial material myxin comprises the following steps: amplification ofphzA2The upstream fragment and the downstream fragment of the gene are connected to a suicide vector pEX18Gm after being treated by restriction enzyme, the recombinant vector is transformed into Pseudomonas aeruginosa TC3 through an amphiphilic joining mode, and the recombinant vector is finally screened out after two homologous recombinationphzA2Deletion mutant designated deltaphzA2。
Specifically, the method for gene knockout by homologous recombination specifically comprises the following steps:
amplification by PCRphzA2The sequences of the upstream and downstream fragments phzA2-S and phzA2-x of the gene are respectively shown as SEQ ID NO.2 and SEQ ID NO.3, the upstream and downstream fragments are subjected to linearization treatment by restriction enzymes and then are connected to a pEX18Gm suicide vector to obtain a homologous recombinant vector, and the homologous recombinant vector is transferred into escherichia coli S17-1 lambda by heat shockpirIn the method, the recombinant vector constructed in the method is transferred into TC3 for amphipathic conjugation, a primary exchanger is obtained through gentamicin and ampicillin dual-resistance plate screening, a secondary exchanger is obtained through sucrose plate screening, and finally the recombinant vector is obtained through PCR verificationphzA2Deletion mutant deltaphzA2。
The chassis strain is applied to the fermentation production of phenazine-1, 6-dicarboxylic acid.
The invention obtains the pseudomonas aeruginosa which has a certain tolerance capacity to the myxin and possibly has a similar efflux system with the original bacterial strain OH13 of the myxin through screeningPseudomonas chlororaphis) TC3, knocking out phenazine synthesis related genes by using a homologous recombination method through genetic engineering modification on the basis of the strainphzA2Obtaining strain deltaphzA2The strain can generate a phenazine-1, 6-dicarboxylic acid PDC which is a synthesized precursor substance of myxin, and lays a foundation for subsequent heterologous biosynthesis of myxin in the strain.
The method for testing the tolerance of the strain to myxin comprises the following steps:
after myxin is dissolved by DMSO, mother liquor with the concentration of 1000 mug/mL is prepared, NA plates (the final concentration is 0, 2,4 and 8 mug/mL) containing pure myxin with different concentrations are prepared, 2 mug of TC3 bacteria liquid is spotted on the plates, the original producing bacteria OH13 of the myxin is used as a control, and colony growth condition is observed after 24 h, so that TC3 has certain tolerance to the myxin is judged.
Advantageous effects
Phenazine-1-carboxylic acid (PCA) and phenazine-1, 6-dicarboxylic acid (PDC) are synthetic substrates or precursors of two major natural phenazine compounds, but Pseudomonas itself only produces phenazine-1-carboxylic acid (PCA) and does not produce phenazine-1, 6-dicarboxylic acid (PDC), a synthetic substrate for myxin. Compared with the prior art, the chassis strain suitable for the heterologous biosynthesis of the antibacterial substance myxin, the construction method and the application thereof, provided by the invention, are used for obtaining the Pseudomonas aeruginosa which has a certain tolerance capacity to myxin and possibly has a similar excretion system with the original bacterial strain OH13 of the myxin through screeningPseudomonas chlororaphis) TC3, knocking out phenazine synthesis related genes by using a homologous recombination method through genetic engineering modification on the basis of the strainphzA2Obtaining strain deltaphzA2Engineering strain delta obtained through the gene editingphzA2Not only PCA but also PDC can be produced, providing a suitable chassis strain for subsequent further acquisition of myxin or other active substances based on PDC as precursor by heterologous biosynthesis.
Drawings
FIG. 1 shows that Pseudomonas aeruginosa TC3 has a certain tolerance to myxin;
FIG. 2 is a schematic view ofphzA2Electrophoresis verification diagram of gene deletion mutant;
FIG. 3 shows deltaphzA2Generating a liquid chromatography detection chart of the PDC;
FIG. 4A is a signal peak for a material having a molecular weight of 268;
FIG. 4B is the molecular weight of the 9.1min signal peak.
Description of the embodiments
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The escherichia coli strain dh5α used in the following examples was purchased from the family of the organisms (Tsingke Biotechnology co., ltd.). Pseudomonas aeruginosaPseudomonas chlororaphis) TC3 was deposited in China general microbiological culture Collection center CGMCC (China general microbiological culture Collection center) at about 05 and 19 months 2022, and the accession number is: CGMCC No.24936, preservation address: the institute of microorganisms of national academy of sciences of China, no.1, no.3, north Chen West Lu, the Korean region of Beijing. The vector pEX18GM is also a commercially available product. Primers used in the examples were synthesized by the same organism (Tsingke Biotechnology co., ltd.). The culture medium LB and the culture medium NA used in the examples are common in the art, and thus are not described in detail.
EXAMPLE 1 Pseudomonas aeruginosaPseudomonas chlororaphis) Detection of TC3 tolerance to myxin
Dissolving pure myxin in DMSO, preparing mother solution with concentration of 1000 mug/mL, adding 0, 40, 80 and 160 mug mother solution into 20 mL NA culture medium, preparing NA plate containing 0, 2,4 and 8 mug/mL myxin, spotting 2 mug TC3 fresh bacteria solution on plates with different concentration myxin, simultaneously taking original producing bacteria OH13 of myxin as reference, placing the plates in a 28 ℃ incubator for standing culture for 24 h, and observing colony growth.
As shown in FIG. 1, TC3 grew well on NA plates containing 4. Mu.g/mL myxin and started to be inhibited on NA plates containing 8. Mu.g/mL myxin, indicating that TC3 strain had a certain tolerance to myxin.
phzA2The specific scheme is as follows:
the primers phzA2-F1/R1 and phzA2-F2/R2 are used for amplifying the upstream and downstream fragments phzA2-s and phzA2-x of the phzA2 gene respectively, and the primer sequences are as follows:
phzA2-F1：CGGAATTCGCACTAACGGCAAAGATCGC (underlined indicates EcoRI cleavage site)
phzA2-R1：GCTCTAGACGTTAGCGGTTAGTTGTTGC (underlined indicates XbaI cleavage site)
phzA2-F2：GCTCTAGAGGCAAAGTAAAACGCAATCG (underlined indicates XbaI cleavage site)
phzA2-R2：CCCAAGCTTTGCGTTCGTTGTTGTTGGAG (HindIII cleavage site is underlined)
The upstream fragment of phzA2-s is treated by restriction enzymes EcoRI and XbaI, the downstream fragment of phzA2-x is treated by XbaI and HindIII, the fragment is connected with a T4 ligase to a suicide vector pEX18Gm treated by EcoRI and HindIII, the fragment is transformed into E.coli DH5 alpha-competent cells by heat shock, and the correct clone is obtained by PCR screening with the primers phzA2-F1/R2phzA2The recombinant vector phzA2-pEX18 with the gene knocked out.
The recombinant vector phzA2-pEX18 is transformed into the escherichia coli S17-1 lambda by heat shockpirIn S17-1 lambda containing recombinant vectorpirAfter the bacterial strain is subjected to co-culture with pseudomonas aeruginosa TC3 to generate amphipathic conjugation, the bacterial strain is coated on an LB solid plate containing 150 mug/mL gentamicin and 100 mug/mL ampicillin, and is subjected to stationary culture at 28 ℃ for 48 h, and colonies growing on the plate are primary exchangers subjected to primary homologous recombination. The micro-bacteria are picked up by a sterilized toothpick and shake-cultured in 1 mL LB liquid medium at 28 ℃ for 6 h, and coated on an LB solid plate containing 15% sucrose, and then the micro-bacteria are statically cultured at 28 ℃ for 48 h, and a single colony growing on the plate is the secondary exchanger with the second homologous recombination. The secondary exchanger was screened with the primer phzA2-F1/R2, the results are shown in FIG. 2, and the final product without introducing any resistance was obtainedphzA2Deletion mutant deltaphzA2。
In the above example, the Pseudomonas aeruginosa TC3phzA2The gene sequence is shown in SEQ ID NO. 1:
ATGCCTAACAGTGCAACGCAACAACTAACCGCTAACGACACCACTGAACTTCGCCGCAAAAACCGCGCCACGGTCGAGCAATATATGCGGACCAAAGGCCAGGACCGCCTGCGCCGCCATGAACTATTTACCGAAGATGGTACAGGCGGCTTATGGACCACCGACACCGGCGCGCCGATTGTAATCAGTGGCAAAGCCAAGTTGGCCGAACATGCTATTTGGTCACTCAAGTGCTTCCCGGACTGGGAATGGTACAACGTCAAAGTATTTGAAACCGATGACCCTAACCATATCTGGGTCGAGTGCGATGGCCGCGGCAAGATCCTCTTTCCCGGCTATCCGGAAGGCTATTACGAGAACCACTTCCTGCACTCCTTCGAGCTGGAAGACGGCAAAGTAAAACGCAATCGCGAATTCATGAACGTCTTTCAGCAACTGCGCGCCCTGGGTATTCCAGTACCGCAAATCAAACGCGAAGGCATTCCAACTTAA
in the above embodiment, the sequence of the PhzA2-s gene fragment is shown in SEQ ID NO. 2:
GCACTAACGGCAAAGATCGCCTGCGCCGGCATGAACTGTTCACTCAGGATGGCAGTGGCGGTTCCTGGAACACCGAAACCGGCAAACCCCTCGTGTTCCAGGGCCACGCGAAGCTGGCCGCCCTCGGCGCATGGCTGGAGAAGTGCTTTCCAGACTGGCAATGGCACAACGTCCGGGTGTTTGAAACCGATAACCCCAACCACTTCTGGGTGGAGAGCGACGGTCGCGGCAAGACTCTGGTTCCCGGTTATCCCGAAGGTTATTGCGAAAACCATTACATCCATTCTTTCGAACTGGATGACGGCAAGATCACGCAGAGCCGTGAATTCATGAACCCCTTCGAACAACTTCGCGCCTTGGGTATCCCCGTTCCAAGAATCAAGCGTGAAGGTATCCCCGCCTCATAAGCCCCCCCTAATTCATTGGAGATTGAGCATGCCTAACAGTGCAACGCAACAACTAACCGCTAACG
in the above embodiment, the sequence of the PhzA2-x gene fragment is shown in SEQ ID NO. 3:
GGCAAAGTAAAACGCAATCGCGAATTCATGAACGTCTTTCAGCAACTGCGCGCCCTGGGTATTCCAGTACCGCAAATCAAACGCGAAGGCATTCCAACTTAATCCCTCGTGAGAGTGATCGCATCATGGAAGACTTACTGAAACGGGTTTTAAGTTGTGAAGCGTTCCAGCAGCCTCAATGGAGCGAGCCCTCACAATTGCACGACGCGCAGGCCTACCTCAGGGACAGCGCCTCATTGATACGAGTGGAAGACATCCTGGTGCTGCGCGCCACGCTGGCTCGTGTAGCGGCCGGCGAAGCGATGGTCATCCAGTCCGGTGACTGCGCCGAGGACATGGATGAAAGCACTCCCGACCATGTGGCCCGCAAAGCCGCGGTGCTGGACATCCTGGCCGGTACGTTCCGGCTGGTGACCCAGCAACCGGTGGTACGGGTGGGCCGGATTGCCGGGCAGTTTGCCAAGCCGCGCTCCAACAACAACGAACGCA
EXAMPLE 3 engineering Strain deltaphzA2Detection of middle PDC
Wild strain TC3 and engineering strain deltaphzA2Respectively inoculating in NA culture medium, and shaking culturing at 28deg.C with horizontal shaking table 24 h. Extracting metabolite in 2 mL bacteria solution with equal volume of ethyl acetate, blow drying, dissolving with 200 μl methanol, and detecting whether PDC is generated by liquid chromatography.
The results are shown in FIG. 3, delta compared to PDC standardphzA2The strain produced a signal response at the same time, whereas the wild-type strain TC3 did not at the peak time, indicating that the engineered strain deltaphzA2PDC may be produced.
To further verify deltaphzA2If PDC is synthesized, the sample corresponding to the suspected PDC signal in the above detection is subjected to HPLC/MS detection, and as a result, as shown in fig. 4, the cation result shows that there is a signal at a mass-to-charge ratio 269, that is, the molecular weight of the substance is 268, which matches the molecular weight of PDC. In conclusion, the engineering strain delta constructed by the inventionphzA2PDC may be produced.
In the foregoing, the protection scope of the present invention is not limited to the preferred embodiments of the present invention, and any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention disclosed in the present invention fall within the protection scope of the present invention.
Claims (4)
1. A chassis strain suitable for heterologous biosynthesis of antibacterial substances myxin is characterized in that the chassis strain adopts pseudomonas aeruginosaPseudomonas chlororaphis) TC3 as an original strain and knocks out phenazine synthesis genes by a homologous recombination methodphzA2The starting strain Pseudomonas aeruginosaPseudomonas chlororaphis) TC3 was deposited in China general microbiological culture Collection center CGMCC (China general microbiological culture Collection center) at about 05 and 19 months 2022, and the accession number is: CGMCC No.24936, preservation address: the institute of microorganisms of national academy of sciences of China, no.1, no.3, north Chen West Lu, the Korean region of Beijing.
2. A chassis strain suitable for heterologous biosynthesis of antibacterial substances myxin according to claim 1, wherein the said strain is encodedphzA2The nucleotide sequence of the gene is shown as SEQ ID No. 1.
3. A method for constructing a chassis strain suitable for heterologous biosynthesis of an antibacterial substance myxin according to claim 1, characterized in that: amplification ofphzA2The upstream fragment and the downstream fragment of the gene are connected to a suicide vector pEX18Gm after being treated by restriction enzyme, and the recombinant vector is transformed into Pseudomonas aeruginosa TC3 by an amphiphilic conjugation mode and subjected to homologous recombination twiceGroup, finally screen outphzA2Deletion mutant designated deltaphzA2。
4. Use of a chassis strain according to any of claims 1-3 for the fermentative production of phenazine-1, 6-dicarboxylic acid.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2022112120988 | 2022-09-30 |
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| CN117778280A true CN117778280A (en) | 2024-03-29 |
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