CN115820676B - Application of Pa2643 gene in regulating and controlling yield of pantoea agglomerans synthesized moxidectin A - Google Patents
Application of Pa2643 gene in regulating and controlling yield of pantoea agglomerans synthesized moxidectin A Download PDFInfo
- Publication number
- CN115820676B CN115820676B CN202211132397.0A CN202211132397A CN115820676B CN 115820676 B CN115820676 B CN 115820676B CN 202211132397 A CN202211132397 A CN 202211132397A CN 115820676 B CN115820676 B CN 115820676B
- Authority
- CN
- China
- Prior art keywords
- pantoea agglomerans
- gene
- moxidectin
- zju23
- yield
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 60
- 241000588912 Pantoea agglomerans Species 0.000 title claims abstract description 58
- YZBLFMPOMVTDJY-CBYMMZEQSA-N moxidectin Chemical compound O1[C@H](C(\C)=C\C(C)C)[C@@H](C)C(=N/OC)\C[C@@]11O[C@H](C\C=C(C)\C[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 YZBLFMPOMVTDJY-CBYMMZEQSA-N 0.000 title claims abstract description 34
- 229960004816 moxidectin Drugs 0.000 title claims abstract description 34
- 230000001276 controlling effect Effects 0.000 title claims abstract description 8
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 8
- 238000000855 fermentation Methods 0.000 claims abstract description 29
- 230000004151 fermentation Effects 0.000 claims abstract description 29
- 241000894006 Bacteria Species 0.000 claims abstract description 25
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 239000001963 growth medium Substances 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 5
- 238000009630 liquid culture Methods 0.000 claims abstract description 3
- 239000002609 medium Substances 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- 238000004519 manufacturing process Methods 0.000 claims description 11
- 238000010353 genetic engineering Methods 0.000 claims description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 238000004321 preservation Methods 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000000243 solution Substances 0.000 description 12
- 240000000220 Panda oleosa Species 0.000 description 10
- 235000016496 Panda oleosa Nutrition 0.000 description 10
- 239000012634 fragment Substances 0.000 description 10
- 238000003209 gene knockout Methods 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 6
- 238000011144 upstream manufacturing Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 244000025254 Cannabis sativa Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- PERZMHJGZKHNGU-JGYWJTCASA-N bambermycin Chemical compound O([C@H]1[C@H](NC(C)=O)[C@@H](O)[C@@H]([C@H](O1)CO[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@@H]1O[C@@H]([C@H]([C@H](O)[C@H]1NC(C)=O)O[C@H]1[C@@H]([C@@H](O)[C@@H](O)[C@H](O1)C(=O)NC=1C(CCC=1O)=O)O)C)[C@H]1[C@@H](OP(O)(=O)OC[C@@H](OC\C=C(/C)CC\C=C\C(C)(C)CCC(=C)C\C=C(/C)CCC=C(C)C)C(O)=O)O[C@H](C(O)=O)[C@@](C)(O)[C@@H]1OC(N)=O PERZMHJGZKHNGU-JGYWJTCASA-N 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 241000223195 Fusarium graminearum Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 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
- 229960000723 ampicillin Drugs 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000030279 gene silencing Effects 0.000 description 2
- 238000012226 gene silencing method Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 229930000044 secondary metabolite Natural products 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 description 1
- RQOCXCFLRBRBCS-UHFFFAOYSA-N (22E)-cholesta-5,7,22-trien-3beta-ol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CCC(C)C)CCC33)C)C3=CC=C21 RQOCXCFLRBRBCS-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241001225321 Aspergillus fumigatus Species 0.000 description 1
- 241000123650 Botrytis cinerea Species 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- DNVPQKQSNYMLRS-NXVQYWJNSA-N Ergosterol Natural products CC(C)[C@@H](C)C=C[C@H](C)[C@H]1CC[C@H]2C3=CC=C4C[C@@H](O)CC[C@]4(C)[C@@H]3CC[C@]12C DNVPQKQSNYMLRS-NXVQYWJNSA-N 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 229940091771 aspergillus fumigatus Drugs 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- DNVPQKQSNYMLRS-SOWFXMKYSA-N ergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H](CC[C@]3([C@H]([C@H](C)/C=C/[C@@H](C)C(C)C)CC[C@H]33)C)C3=CC=C21 DNVPQKQSNYMLRS-SOWFXMKYSA-N 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 244000000004 fungal plant pathogen Species 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 108091008053 gene clusters Proteins 0.000 description 1
- 108700024565 herbicolin A Proteins 0.000 description 1
- GMKUYPOKOFFZTR-KJRGEGHNSA-N herbicolin a Chemical compound O1C(CO)C(O)C(O)C(O)C1OC(CCCCCCCCCCC)CC(=O)N\C(=C\C)C(=O)NC1C(C)OC(=O)C(CCCN=C(N)N)NC(=O)C(C(C)O)N(C)C(=O)CNC(=O)C(CCC(N)=O)NC(=O)CNC(=O)C(CC(C)C)NC(=O)C(C(C)O)NC1=O GMKUYPOKOFFZTR-KJRGEGHNSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 108091008023 transcriptional regulators Proteins 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an application of Pa2643 gene in regulating and controlling the yield of pantoea agglomerans synthesized moxidectin A, wherein the nucleotide sequence of the Pa2643 gene is shown as SEQ ID No. 1; the application way is to improve the yield of the pantoea agglomerans synthesized moxidectin A by disrupting the function of the Pa2643 gene. Based on the application, the invention obtains the pantoea agglomerans engineering bacteria ZJU23-Pa2643 capable of producing the moxidectin A, the pantoea agglomerans engineering bacteria ZJU23-Pa2643 is obtained by knocking out Pa2643 genes in the pantoea agglomerans ZJU23, and the pantoea agglomerans engineering bacteria ZJU23-Pa2643 is inoculated into KB liquid culture medium for fermentation for 72 hours, so that the yield of the moxidectin A in fermentation liquid is up to 197.9mg/L and is 2.28 times of that of the wild pantoea agglomerans ZJU 23.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to application of Pa2643 gene in controlling the yield of pantoea agglomerans synthesized moxidectin A.
Background
The moenomycin A (Herbicolin A) is a secondary metabolite produced by pantoea agglomerans (Pantoea agglomerans) ZJU 23. The grass eumycin A has inhibiting effect on plant pathogenic fungi such as rice blast germ, fusarium graminearum, botrytis cinerea and the like, and human conditional pathogenic bacteria such as candida albicans, aspergillus fumigatus and the like. The grass eumycin A (structural formula is shown as formula (1)) has a unique antibacterial mechanism, can damage the structure of lipid rafts on a membrane by combining ergosterol on the cell membrane of fusarium graminearum, thereby exerting antibacterial activity, and can be used as a good biological source pesticide for biological control. The moenomycin A has many advantages, but its production cost is enormous, so that increasing its yield becomes a core problem for reducing its production cost.
At present, the method for producing the moxidectin A is mainly a microbial fermentation method, and the chemical synthesis method for obtaining the moxidectin A is not reported at present. The microbial fermentation method mainly obtains the moxacillin A through microbial fermentation, and has the advantages of relatively simple process, environment friendliness, mild condition, low cost and the like.
However, existing biological methods still have certain defects, wherein the low yield is the most important defect for preventing the industrial process of the microbial fermentation method. Therefore, it is highly desirable to find a microorganism which can produce high yields of Oenomycin A to overcome the above-mentioned drawbacks.
At present, methods for improving the yield of secondary metabolites of bacteria mainly comprise optimizing fermentation conditions, screening high-yield strains by gene mutation, metabolic engineering and the like. The genes involved in the biosynthesis of moxidectin A are clustered on the genome of Pantoea agglomerans ZJU 23. To date, few studies have been conducted on transcriptional regulators and the regulatory mechanisms involved in the grass eumycin A biosynthetic pathway.
Disclosure of Invention
The invention provides a new application of Pa2643 genes in regulating and controlling the yield of the pantoea agglomerans to synthesize the moxidectin A, and obtains pantoea agglomerans genetic engineering bacteria after the functions of the Pa2643 genes are destroyed according to the new application, and the moxidectin A is produced by fermenting the pantoea agglomerans genetic engineering bacteria, so that the yield of the moxidectin A is remarkably improved.
The specific technical scheme is as follows:
the invention provides application of a Pa2643 gene in regulating and controlling the yield of pantoea agglomerans synthesized moxidectin A, wherein the nucleotide sequence of the Pa2643 gene is shown as SEQ ID No. 1; the application method is to destroy the function of Pa22 gene to improve the yield of the synthetic moxidectin A of the pantoea agglomerans.
In the research of the invention, the gene Pa2643 related to the transcription negative regulation of the moxidectin A gene cluster is mined, and the regulation protein gene is knocked out, so that the yield of moxidectin A can be obviously improved.
The amino acid sequence of the protein encoded by the Pa2643 gene is shown as SEQ ID No.2, the gene encodes 307 amino acids in total and is a transcription regulating factor, and the Pa2643 gene silencing mode can be various, for example: gene knockout, gene silencing, and the like.
Further, the application way is to increase the yield of the synthetic grass eumycin A of the pantoea agglomerans by knocking out the Pa2643 gene of the pantoea agglomerans.
Furthermore, the pantoea agglomerans is pantoea agglomerans (Pantoea agglomerans) ZJU23, the strain preservation number is CGMCC No.16174, and the preservation date is 2018, 7, 30.
The invention also provides a pantoea agglomerans genetic engineering bacterium, which is pantoea agglomerans (Pantoea agglomerans) ZJU23 with Pa2643 gene knocked out; the nucleotide sequence of the Pa2643 gene is shown as SEQ ID No. 1.
Further, the genetically engineered bacterium also comprises plasmid pKD46.
The knockout principle is as follows: the resistant fragment containing 50bp homology arms at the upstream and downstream of Pa2643 gene is transferred into host cell ZJU23, and Pa2643 gene is knocked out by homologous recombination with the help of recombinase expressed by plasmid pKD46.
The invention also provides application of the pantoea agglomerans genetic engineering bacteria in improving the yield of the moxidectin A, wherein the pantoea agglomerans genetic engineering bacteria are as described above.
The invention also provides a method for producing the moxidectin A, which comprises the following steps: inoculating the genetically engineered bacteria of the pantoea agglomerans into a fermentation medium for fermentation to obtain fermentation liquor containing the moxacillin A; separating the fermentation liquor containing the moxidectin A to obtain the moxidectin A;
the genetically engineered bacteria of the Pantoea agglomerans are as described above.
Further, the fermentation temperature is 25-30 ℃ and the rotating speed is 150-200 rpm. Further, the fermentation temperature was 25℃and the rotation speed was 180rpm.
Further, the fermentation medium is KB liquid medium; the KB liquid culture medium comprises 8-12 g/L of peptone, 13-16 g/L of glycerol, 1.2-1.6 g/L of dipotassium hydrogen phosphate and 0.4-0.8 g/L of magnesium sulfate.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers a new application of Pa2643 genes in regulating and controlling the yield of the pantoea agglomerans to synthesize the moxidectin A, obtains a pantoea agglomerans engineering bacterium ZJU23-Pa2643 capable of producing the moxidectin A based on the discovery, wherein the pantoea agglomerans engineering bacterium ZJU23-Pa2643 is obtained by knocking out the Pa2643 genes in the pantoea agglomerans ZJU23, and inoculates the pantoea agglomerans engineering bacterium ZJU23-Pa2643 into KB liquid medium for fermentation for 72 hours, so that the yield of the moxidectin A in a fermentation broth is up to 197.9mg/L and is 2.28 times that of the wild pantoea agglomerans ZJU 23.
Drawings
FIG. 1 is a schematic diagram of the knockout principle of Pa2643 gene;
wherein Pa2643-F, pa2643-R is a primer containing 50bp homology arms at the upstream and downstream of Pa2643 gene and used for amplifying the sequence of the kana resistance gene; pa2643-ID-F, pa2643-ID-R is a primer for verifying whether the Pa2643 gene is knocked out, the primer is respectively designed on the upstream of a 50bp homology arm and on a kana resistance gene fragment, and if the Pa2643 gene is knocked out, a band with the size of 762bp can be amplified; pa2643-N-F, pa2643-N-R is the internal sequence of the Pa2643 gene, if the Pa2643 gene is not knocked out, a band with 834bp can be amplified, so that the Pa2643 gene knocked out mutant cannot amplify the band, and the ZJU23 wild type strain can amplify the band with the corresponding size; km is the sequence of the kana resistance gene.
FIG. 2 is a colony PCR identification chart of Pa2643 gene knockout strain;
wherein ID represents PCR verification by using the primer Pa2643-ID-F, pa2643-ID-R, and IN represents PCR verification by using the primer Pa2643-N-F, pa-2643-N-R. Pa2643-1 is the band of Pa2643 gene knockout strain 1, pa2643-2 is the band of Pa2643 gene knockout strain 2, pa2643-3 is the band of Pa2643 gene knockout strain 3, and three strains are different transformants; ZJU23 is a PCR amplified band of Pantoea agglomerans ZJU23; positive control was used as a Negative control, i.e., no DNA template was added during PCR, and Marker was a 250bp DNA Ladder purchased from Takara, used to indicate the size of the running strip.
FIG. 3 is a schematic diagram showing fermentation yields of the Pantoea agglomerans genetic engineering bacteria ZJU23-Pa2643 and the moxidectin A in the Pantoea agglomerans ZJU23;
wherein 23WT is Pantoea agglomerans ZJU23, ZJU23-Pa2643 is Pantoea agglomerans engineering bacteria ZJU23-Pa2643.
Detailed Description
The invention will be further described with reference to the following examples, which are given by way of illustration only, but the scope of the invention is not limited thereto.
The media and reagents involved in the following examples were as follows:
LB liquid medium: 10g/L of tryptone, 5g/L of yeast powder and 10g/L of sodium chloride. LB solid medium: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of sodium chloride and 15g/L of agar.
KB liquid medium: 10g/L peptone, 15g/L glycerol, 1.5g/L dipotassium hydrogen phosphate and 0.6g/L magnesium sulfate.
The detection method involved in the following examples is as follows:
determination of the content of moenomycin A: after the fermentation broth was obtained, the production of moxidectin a in the fermentation broth was determined by using High Performance Liquid Chromatography (HPLC). The fermentation broth is centrifuged, filtered, and the thalli and other impurities are removed for HPLC determination. The test was carried out using a ZorBax RX C-18 250x 4.6mm column at 40℃with a sample loading of 10. Mu.L and an ultraviolet absorption peak of 210nm. The mobile phase is: solution A (methanol with 0.1% formic acid) and solution B (deionized water with 0.1% phosphoric acid). The sample injection procedure is as follows: 0-30min, 30-90% A;30-40min,90% A-100% A, 100% total ratio of solution A and solution B, and flow rate of 1mL/min. The peak time was about 25.6 minutes with the reference to the moxacillin a standard.
EXAMPLE 1 construction of Pantoea agglomerans engineering bacterium ZJU23-Pa2643
The method comprises the following specific steps:
step one: amplification of the Cana resistance Gene fragment containing the 50bp homology arm upstream and downstream of the Pa2643 gene.
Using plasmid plvc-Km as a template, performing PCR reaction to amplify a resistance fragment by using Pa2643-F, pa2643-R primer, and recovering the corresponding fragment by running gel, wherein the sequence of the kana resistance gene is shown as SEQ ID NO. 3; table 1 lists the primer sequences used in the gene knockout process.
TABLE 1 primer sequences used in Pa2643 Gene knockout procedure
Wherein, the primer Pa2643-F, pa2643-R is used for amplifying the resistance gene fragment, and the primer Pa2643-ID-F, pa2643-ID-R, pa2643-N-F, pa2643-N-R is used for verifying whether the gene is knocked out. Pa2643-F, pa2643-R is a primer containing 50bp homology arms at the upstream and downstream of Pa2643 gene for amplifying the sequence of the kana resistance gene; pa2643-ID-F, pa2643-ID-R is a primer for verifying whether the Pa2643 gene is knocked out, the primer is respectively designed on the upstream of a 50bp homology arm and on a kana resistance gene fragment, and if the Pa2643 gene is knocked out, a band with the size of 762bp can be amplified; pa2643-N-F, pa2643-N-R is the internal sequence of the Pa2643 gene, if the Pa2643 gene is not knocked out, a band with 834bp can be amplified, so that the Pa2643 gene knocked out mutant cannot amplify the band, and the ZJU23 wild type strain can amplify the band with the corresponding size.
Step two: construction of a Pantoea agglomerans genetic engineering bacterium ZJU23/pKD46 with Pa2643 gene knocked out.
The homologous helper plasmid pKD46 selected in this embodiment not only encodes three protein factors of the lambda-red recombination system Exo, bet and Gam, but also is induced to express L-arabinose and contains ampicillin resistance gene selection markers.
First, heat shock competent cells of Pantoea agglomerans ZJU23 were prepared. Picking newly activated ZJU23 single colony from a solid culture medium plate, inoculating the newly activated ZJU23 single colony into 3-5mL of LB culture solution, culturing at 30 ℃ overnight until the late logarithmic growth phase, inoculating the bacterial solution into 100mL of LB according to the ratio of 1:100, and culturing at 30 ℃ for 3-5 h in a shaking way until the bacterial solution reaches OD 600 About 0.5; the culture was transferred to a 50mL centrifuge tube, placed on ice for 30 minutes, and then centrifuged at 4000rpm for 10 minutes at 4 ℃; discarding supernatant, pre-cooling0.1M CaCl of (C) 2 10mL of the solution was gently suspended, and after 30 minutes of incubation on ice, the cells were centrifuged at 4000rpm at 4℃for 5 minutes; the supernatant was discarded and 4mL of pre-chilled 0.1M CaCl containing 15% glycerol was added 2 The solution is used for suspending cells gently, and the competent cell suspension is obtained after the cells are placed on ice for a few minutes; subsequently, 10. Mu.L of plasmid pKD46 was added to 100. Mu.L of competent cell suspension, and after standing for 30 minutes, it was heat-shocked at 42℃for 45s, 800. Mu.L of fresh LB liquid medium was immediately added thereto, and shaking culture was performed at 30℃and 180rpm for 1 hour. And (5) centrifuging the bacterial liquid at 5000rpm for 3min to collect bacterial cells. Resuspended in 100. Mu.L of LB liquid medium and then plated in LB solid medium with a final ampicillin concentration of 100mg/L in an incubator at 30℃overnight. Picking transformed single colony, inoculating into 3mL LB culture medium (containing 100mg/L Amp), shake culturing at 30deg.C for 12-16h or overnight, and preserving at-80deg.C;
step three: the kana resistance gene fragment was electric shock transferred into ZJU23/pKD46 and screened for verification mutants.
Picking ZJU23/pKD46 single colony from LB plate in 3mL LB culture solution, culturing at 30deg.C and 180rpm overnight to obtain seed solution; the seed solution was then transferred to 100mL of LB liquid medium (arabinose was added to the medium at a final concentration of 10 mM) at 1:100, and cultured at 180rpm at 30℃for about 5 hours (OD 600 About 1.0); transferring the culture into a 50mL centrifuge tube, placing on ice for 30 minutes, centrifuging at 4 ℃ and 4000rpm, and washing with precooled deionized water for 3 times to obtain competent cell suspension; then the kana resistance gene fragment containing 50bp target gene homology arm was shocked into competence, using a 2mm cuvette of Bio-Rad for shocking with the following parameters: the voltage is 2.5kV, and the electric shock time is 4.0-5.5ms; after the electrotransformation was completed, 800. Mu.L of liquid LB medium was quickly added to the electrotransformation cup. Transferring the bacterial liquid from the electric shock cup into a 2mL centrifuge tube, and recovering and culturing at 30 ℃ and 180rpm for about 1 hour to ensure that the kana resistance fragment and a target gene of a host strain undergo homologous recombination; and screening by using the kana resistance, and selecting single colonies for PCR verification to obtain the high-yield strain ZJU23-Pa2643 of the moenomycin A.
Example 2 production of moxidectin A
The method comprises the following specific steps:
activating the wild strain ZJU23 and the gene knockout mutant strain ZJU23-Pa2643 on an LB solid medium, and inoculating the activated strain into an LB liquid medium to culture for 12 hours at 30 ℃ and a rotating speed of 180 rpm; the inoculated culture medium was inoculated at an inoculum size of 1:1000 and cultured at 25℃and 180rpm for 72 hours, followed by collection of the fermentation broth.
After the fermentation broth was obtained, the production of moxidectin a was determined by using a High Performance Liquid Chromatography (HPLC) pair.
The results of the measurement of the yield of the moxacillin A are shown in FIG. 3. Compared with the wild strain ZJU23, the mutant strain 23 has obviously improved mutant strain ZJU23-Pa2643. The indoor shake flask fermentation yield reaches 197.9mg/L, which is 2.28 times of the wild strain.
Claims (7)
- The application of the Pa2643 gene in regulating and controlling the yield of the pantoea agglomerans synthesized moxibusin A is characterized in that the nucleotide sequence of the Pa2643 gene is shown as SEQ ID No. 1; the application method is that the Pa2643 gene of the Pantoea agglomerans (Pantoea agglomerans) ZJU23 is knocked out to improve the yield of the Pantoea agglomerans synthetic moxidectin A;the preservation number of the pantoea agglomerans (Pantoea agglomerans) ZJU23 is CGMCC No.16174, and the preservation date is 2018, 7, 30.
- 2. The genetic engineering bacterium of the pantoea agglomerans is pantoea agglomerans (Pantoea agglomerans) ZJU23 with Pa2643 gene knocked out, the strain preservation number is CGMCC No.16174, and the preservation date is 2018, 7 months and 30 days; the nucleotide sequence of the Pa2643 gene is shown as SEQ ID No. 1.
- 3. The genetically engineered bacteria of pantoea agglomerans of claim 2, further comprising plasmid pKD46.
- 4. The application of the genetically engineered bacteria of the pantoea agglomerans in improving the yield of the moxidectin A, which is characterized in that the genetically engineered bacteria of the pantoea agglomerans are as claimed in claim 3.
- 5. A method for producing moxidectin a, comprising: inoculating the pantoea agglomerans engineering bacteria into a fermentation medium for fermentation to obtain fermentation liquor containing the moxacillin A; separating the fermentation liquor containing the moxidectin A to obtain the moxidectin A; the genetically engineered bacteria of Pantoea agglomerans according to any one of claims 3 to 4.
- 6. The method for producing moxacillin a according to claim 5, wherein the fermentation temperature is 25 to 30 ℃ and the rotation speed is 150 to 200rpm.
- 7. The method for producing moxidectin a of claim 5, wherein said fermentation medium is a KB liquid medium; the KB liquid culture medium comprises 8-12 g/L of peptone, 13-16 g/L of glycerol, 1.2-1.6 g/L of dipotassium hydrogen phosphate and 0.4-0.8 g/L of magnesium sulfate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211132397.0A CN115820676B (en) | 2022-09-14 | 2022-09-14 | Application of Pa2643 gene in regulating and controlling yield of pantoea agglomerans synthesized moxidectin A |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211132397.0A CN115820676B (en) | 2022-09-14 | 2022-09-14 | Application of Pa2643 gene in regulating and controlling yield of pantoea agglomerans synthesized moxidectin A |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115820676A CN115820676A (en) | 2023-03-21 |
| CN115820676B true CN115820676B (en) | 2024-04-09 |
Family
ID=85523682
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211132397.0A Active CN115820676B (en) | 2022-09-14 | 2022-09-14 | Application of Pa2643 gene in regulating and controlling yield of pantoea agglomerans synthesized moxidectin A |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115820676B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110713951A (en) * | 2019-11-11 | 2020-01-21 | 浙江大学 | Pantoea agglomerans strain and application thereof |
| CN113016818A (en) * | 2021-03-29 | 2021-06-25 | 浙江大学 | Application of alopecuromycin A in preparing medicament for preventing and treating wheat scab |
| CN113528550A (en) * | 2021-06-08 | 2021-10-22 | 浙江大学 | Biosynthesis gene cluster of oxalomacin and application thereof |
| CN115011614A (en) * | 2022-03-16 | 2022-09-06 | 浙江大学 | Application of Pa22 gene as negative regulatory factor in improving yield of Pantoea agglomerans in synthesis of herbicidin A |
-
2022
- 2022-09-14 CN CN202211132397.0A patent/CN115820676B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110713951A (en) * | 2019-11-11 | 2020-01-21 | 浙江大学 | Pantoea agglomerans strain and application thereof |
| CN113016818A (en) * | 2021-03-29 | 2021-06-25 | 浙江大学 | Application of alopecuromycin A in preparing medicament for preventing and treating wheat scab |
| CN113528550A (en) * | 2021-06-08 | 2021-10-22 | 浙江大学 | Biosynthesis gene cluster of oxalomacin and application thereof |
| CN115011614A (en) * | 2022-03-16 | 2022-09-06 | 浙江大学 | Application of Pa22 gene as negative regulatory factor in improving yield of Pantoea agglomerans in synthesis of herbicidin A |
Non-Patent Citations (1)
| Title |
|---|
| Enhancement of herbicolin A production by integrated fermentation optimization and strain engineering in Pantoea agglomerans ZJU23;Hongkai Wang等;Microb Cell Fact;第22卷(第1期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115820676A (en) | 2023-03-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2021185039A1 (en) | Method for constructing ergothioneine-producing strain | |
| CN110241061B (en) | Method for improving synthesis capacity of lactobacillus brevis gamma-aminobutyric acid and application thereof | |
| WO2009117843A1 (en) | An amidase gene knock-out engineered strain for nitrile hydratase production, its construction and application | |
| CN110699394B (en) | Bioconversion method for producing 1, 5-pentanediamine | |
| CN110951667B (en) | Fenogen element high-yield strain LPB-18N and breeding and application thereof | |
| US20160289690A1 (en) | Mortierella alpina recombinant gene expression system and construction method and use thereof | |
| JP2008541775A (en) | Modified microorganism with inactivated lactate dehydrogenase gene | |
| KR101581504B1 (en) | Recombinant microorganism having enhanced butanediol producing ability and method for producing butanediol using the same | |
| CN115011614B (en) | Application of Pa22 gene as negative regulatory factor in improving yield of pantoea agglomerans synthesized moxidectin A | |
| CN113461789B (en) | LysR family transcription regulation protein derived from Burkholderia, gene and application | |
| CN109486688B (en) | Trichoderma reesei genetic engineering bacterium and preparation method and application thereof | |
| CN104630100A (en) | Reconstructed Klebsiella pneumoniae and application of reconstructed Klebsiella pneumoniae in production of R-acetoin | |
| CN112375723B (en) | Engineering bacteria for producing maleic acid and construction method and application thereof | |
| CN115820676B (en) | Application of Pa2643 gene in regulating and controlling yield of pantoea agglomerans synthesized moxidectin A | |
| CN115948402A (en) | Recombinant Shewanella capable of producing 5-aminolevulinic acid and application thereof | |
| CN113249240B (en) | Saccharomyces cerevisiae for high yield of hydroxytyrosol and construction method thereof | |
| CN111041020B (en) | Isocitrate lyase mutant, mutant gene and application thereof in preparation of vitamin B12In (1) | |
| CN110904079B (en) | β -fructofuranosidase mutant, mutant gene and application thereof in preparation of vitamin B12In (1) | |
| CN111647544A (en) | Genetic engineering strain for producing demethylated aureomycin and demethylated tetracycline and construction method thereof | |
| CN114381417B (en) | Method for improving tolerance of corynebacterium glutamicum to inhibitor | |
| CN113913448B (en) | Method for improving yield of pyrroloquinoline quinone of methylotrophic bacteria and application | |
| CN114806992B (en) | RSH over-expression genetic engineering amylase streptomyces chromogenes and method for improving fermentation yield of toyocamycin | |
| CN114806982B (en) | Modified 1, 3-propanediol producing strain and application thereof | |
| CN115838644A (en) | Microorganism and method for improving fermentation yield of hydrophobic compound of microorganism | |
| CN116064357B (en) | Improved strain and improvement method for effectively improving Fcl29 yield |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |