WO2018036479A1 - 高产吩嗪-1-甲酰胺的基因工程菌株及其构建方法和用途 - Google Patents

高产吩嗪-1-甲酰胺的基因工程菌株及其构建方法和用途 Download PDF

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WO2018036479A1
WO2018036479A1 PCT/CN2017/098480 CN2017098480W WO2018036479A1 WO 2018036479 A1 WO2018036479 A1 WO 2018036479A1 CN 2017098480 W CN2017098480 W CN 2017098480W WO 2018036479 A1 WO2018036479 A1 WO 2018036479A1
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gene
genetically engineered
strain
phenazine
carboxamide
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French (fr)
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彭华松
张雪洪
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上海交通大学
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Priority claimed from CN201610795467.9A external-priority patent/CN107043730A/zh
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/78—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Pseudomonas
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas

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  • the invention belongs to the field of genetic engineering, and relates to a pseudomonas genetic engineering strain, a construction method thereof and a use thereof, in particular to a genetically engineered strain with high yield of phenazine-1-carboxamide, a construction method thereof and use thereof.
  • PCN phenazine-1-carboxamide
  • PCA phenazine-1-carboxamide
  • the object of the present invention is to provide a genetically engineered strain with high yield of phenazine-1-carboxamide and a construction method thereof, which overcomes the low yield and low efficiency of producing phenazine-1-carboxamide by the existing strain.
  • a defect provides a genetically engineered strain for the production of phenazine-1-carboxamide and its use.
  • the present invention relates to a genetically engineered strain of high-yield phenazine-1-carboxamide, which specifically knocks out the genome of Pseudomonas chlororaphis HT66 CCTCC NO: M 2013467 and its derivatives a genetically engineered strain obtained by one or two genes of the lon gene and the parS gene;
  • a genetically engineered strain is obtained by knocking out one or two genes of the Pseudomonas chlororaphis HT66 CCTCC NO: M 2013467rpeA, the psrA single mutant or the lon gene of the double mutant genome, or the parS gene;
  • the single mutant strain is HT66 ⁇ rpeA, HT66 ⁇ psrA, and the double mutant strain is HT66 ⁇ rpeA ⁇ psrA;
  • the base sequence of the lon gene is as shown in SEQ ID NO. 1; the base sequence of the parS gene is shown in SEQ ID NO. 2, and the base sequence of the pykA gene is SEQ ID NO. As shown in .11, the amino acid sequence of the corresponding protein of the pykA gene is shown in SEQ ID NO.
  • the genetically engineered strain comprises HT66 ⁇ lon, HT66 ⁇ parS, HT66 ⁇ lon ⁇ parS, HT66 ⁇ lon ⁇ rpeA, HT66 ⁇ parS ⁇ rpeA, HT66 ⁇ lon ⁇ psrA, HT66 ⁇ psrA ⁇ parS, HT66 ⁇ lon ⁇ parS ⁇ rpeA, HT66 ⁇ lon ⁇ parS ⁇ psrA, HT66 ⁇ lon ⁇ psrA ⁇ rpeA, HT66 ⁇ parS ⁇ psrA ⁇ rpeA, HT66 ⁇ lon ⁇ parS ⁇ psrA ⁇ rpeA, HT66 ⁇ pykA.
  • the present invention provides a method for constructing a genetically engineered strain according to the aforementioned high-yield phenazine-1-carboxamide, which specifically comprises: knocking out Pseudomonas chlororaphis HT66 CCTCC NO: M2013467 and The lon gene of the derivative genome, one gene or two genes of the parS gene;
  • a genetically engineered strain is obtained by knocking out one or two genes of the Pseudomonas chlororaphis HT66 CCTCC NO: M 2013467rpeA, the psrA single mutant or the lon gene of the double mutant genome, or the parS gene;
  • the single mutant strain is HT66 ⁇ rpeA, HT66 ⁇ psrA, and the double mutant strain is HT66 ⁇ rpeA ⁇ psrA;
  • the pykA gene in the genome of Psedunomonaschlororaphis HT66 CCTCC NO: M2013467 and its derivatives is knocked out.
  • the construction method includes:
  • the method of knocking out the pykA gene is an insertion mutation method or a non-marking knockout method
  • the insertion of the mutation method to knock out the pykA gene includes the following steps:
  • the non-markable knockout pykA gene comprises the following steps:
  • A1 amplifying the upstream and downstream homology arms of the pykA gene
  • A2 fusion PCR method is connected to the upstream and downstream homology arms and inserted into the pK18mobsacB plasmid;
  • the present invention provides a method for synthesizing phenazine-1-carboxamide based on the aforementioned genetically engineered strain, which specifically comprises the steps of: inoculating a seed fermentation broth of the genetically engineered strain into a KB fermentation medium for culturing , you can.
  • the seed fermentation broth is prepared by inoculating the genetically engineered strain in KB medium and culturing at 28° C. and 180 rpm until the OD600 is 0.5 to 2.0.
  • the culture conditions are: 26 to 34 ° C, 100 to 300 rpm, and 24 to 72 hours.
  • the volume ratio of the seed fermentation broth to the KB fermentation medium is (1 to 10): 100.
  • the present invention provides a method for synthesizing phenazine-1-carboxamide based on the aforementioned genetically engineered strain, Specifically, the method comprises the steps of: inoculating the genetically engineered strain in a KB liquid medium;
  • the present invention provides a medium for use in the aforementioned genetically engineered strain comprising the following components in parts by weight:
  • the present invention provides the use of a genetically engineered strain of high yield phenazine-1-carboxamide as described above for the production of phenazine-1-carboxamide.
  • the genetic engineering strain has the following conditions for preparing phenazine-1-carboxamide: aerobic culture; temperature: 26-34 ° C; pH: 6-8; and rotation speed of 100-350 rpm.
  • the present invention has the following beneficial effects:
  • the invention specifically uses Pseudomonas sphaeroides HT66 as a starting strain, and NR66 ⁇ lon, HT66 ⁇ parS, HT66 ⁇ lon ⁇ parS, HT66 ⁇ lon, HT66 ⁇ parS, HT66 ⁇ lon ⁇ parS are constructed by genetic engineering technology from the genome of HT66 strain respectively or simultaneously knocking out lon and parS negative regulatory genes in phenazine synthesis process.
  • the genetically engineered strain of HT66 ⁇ pykA gene greatly increased the yield of phenazine-1-carboxamide (CAS No. 550-89-0, PCN for short) and was more effective due to biological control. Among them, the genetically engineered strain HT66 ⁇ lon ⁇ parS has the best effect.
  • Figure 1 is an electrophoresis pattern of PCR amplification of the lon gene and its upstream and downstream fragments by Pseudomonas aeruginosa HT66 and lon knockout mutants, respectively;
  • marker from left to right are marker, HT66 ⁇ lon, HT66, blank control;
  • FIG. 2 is a growth curve of HT66 ⁇ lon, HT66 ⁇ parS, HT66 ⁇ lon ⁇ parS genetically engineered bacteria
  • Figure 3 is a graph showing the yield of phenazine-1-carboxamide of HT66HT66 ⁇ lon, HT66 ⁇ parS, HT66 ⁇ lon ⁇ parS genetically engineered bacteria;
  • Figure 4 is a growth curve of HT66 ⁇ lon ⁇ rpeA genetically engineered bacteria
  • Figure 5 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ lon ⁇ rpeA genetically engineered bacteria
  • Figure 6 is a growth curve of HT66 ⁇ parS ⁇ rpeA genetically engineered bacteria
  • Figure 7 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ parS ⁇ rpeA genetically engineered bacteria
  • Figure 8 is a growth curve of HT66 ⁇ lon ⁇ parS ⁇ rpeA genetically engineered bacteria
  • Figure 9 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ lon ⁇ parS ⁇ rpeA genetically engineered bacteria
  • Figure 10 is a growth curve of HT66 ⁇ lon ⁇ psrA genetically engineered bacteria
  • Figure 11 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ lon ⁇ psrA genetically engineered bacteria
  • Figure 12 is a growth curve of HT66 ⁇ psrA ⁇ parS genetically engineered bacteria
  • Figure 13 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ psrA ⁇ parS genetically engineered bacteria
  • Figure 14 is a growth curve of HT66 ⁇ lon ⁇ parS ⁇ psrA genetically engineered bacteria
  • Figure 15 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ lon ⁇ parS ⁇ psrA genetically engineered bacteria
  • Figure 16 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ lon ⁇ parS ⁇ psrA ⁇ rpeA genetically engineered bacteria
  • Figure 17 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ lon ⁇ psrA ⁇ rpeA genetically engineered bacteria
  • Figure 18 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ parS ⁇ psrA ⁇ rpeA genetically engineered bacteria
  • Figure 19 is an electrophoresis map of the amplification product of the pykA gene in different strains (L1 is a blank control, L2: the genome of the wild strain HT66 is used as a template, and L3: the genome of the mutant strain HT66 ⁇ pykA is used as a template);
  • Figure 20 is a comparison chart of growth curves of wild strain HT66 and mutant strain HT66 ⁇ pykA;
  • Figure 21 is an electrophoresis map of the amplification product of pykA gene in different strains (L1 is a blank control, L2: using the genome of strain P3 as a template, and L3: using the genome of mutant P3 ⁇ pykA as a template)
  • Figure 22 is a graph showing growth curves of strain P3 and mutant strain P3 ⁇ pykA
  • Figure 23 is a PCN fermentation curve of wild strain HT66 and mutant strain HT66 ⁇ pykA;
  • Figure 24 is a PCN fermentation curve of strain P3 and mutant strain P3 ⁇ pykA.
  • the Pseudomonas choloraphis HT66 of the present invention has been preserved in the China Center for Type Culture Collection (CCTCC).
  • CTCC China Center for Type Culture Collection
  • the address of the depository is: Wuhan University, Wuhan, China, zip code: 430072, date of preservation For: October 12, 2013, the deposit number is: CCTCC NO: M 2013467.
  • the genomic DNA is used as a template to amplify corresponding fragments in the genome.
  • the upstream and downstream PCR products were ligated by fusion PCR, and the fusion PCR product and the pK18mobsacB vector were digested with EcoRI and XbaI, respectively, and the column was recovered, and ligated with T4 ligase to obtain an in vitro mutant plasmid.
  • E. coli S17 was transformed with an in vitro mutant plasmid.
  • the S17 strain carrying the recombinant plasmid was fully activated, inoculated into LB medium containing kanamycin 50 mg/L, and cultured at 37 ° C, 180 rpm for 12 h.
  • the fully activated HT66 strain was inoculated into LB medium and cultured at 28 ° C, 180 rpm for 12 h.
  • the two cells were collected and washed at 5000 rpm.
  • the LB medium was resuspended and mixed in an EP tube with a ratio of HT66:S17 of 3:1, and allowed to stand for 1 h.
  • the mixed bacteria solution was placed in the LB plate.
  • Figure 1 is an electrophoresis pattern of PCR amplification of lon gene and its upstream and downstream fragments by Pseudomonas fluorescens HT66 and lon knockout mutants respectively; wherein, M, Marker; ⁇ , lon gene knockout strain HT66 ⁇ lon; WT, wild strain HT66; N, blank control group.
  • the left primer was the internal primer lon-F/lon-R
  • the right primer was the external primer lon-F1/lon-R2.
  • the genomic DNA is used as a template to amplify corresponding fragments in the genome.
  • the upstream and downstream PCR products were ligated by fusion PCR, and the fusion PCR product and the pK18mobsacB vector were digested with BamHI and HindIII, respectively, and the column was recovered, and ligated with T4 ligase to obtain an in vitro mutant plasmid.
  • the resulting in vitro mutant plasmid was used to transform E. coli S17.
  • the S17 strain carrying the recombinant plasmid was fully activated, inoculated into LB medium containing kanamycin 50 mg/L, and cultured at 37 ° C, 180 rpm for 12 h.
  • the fully activated HT66 strain was inoculated into LB medium and cultured at 28 ° C, 180 rpm for 12 h.
  • the two cells were collected and washed at 5000 rpm.
  • the LB medium was resuspended and mixed in an EP tube with a ratio of HT66:S17 of 3:1, and allowed to stand for 1 h.
  • the mixed bacteria solution was placed in the LB plate.
  • the S17 strain carrying the parS recombinant plasmid was fully activated, inoculated into LB medium containing kanamycin 50 mg/L, and cultured at 37 ° C, 180 rpm for 12 h.
  • the fully activated HT66 ⁇ lon strain was inoculated into LB medium and cultured at 28 ° C, 180 rpm for 12 h.
  • the two cells were collected and washed at 5000 rpm.
  • the LB medium was resuspended and mixed in an EP tube with a ratio of HT66 ⁇ lon:S17 of 3:1. The mixture was allowed to stand for 1 h.
  • the mixed bacteria solution was placed in the LB plate.
  • Example 4 Growth curve measurement of Pseudomonas HT66 and genetically engineered strain HT66 ⁇ lon ⁇ parS
  • the strain was fully activated and inoculated into fresh KB medium, cultured overnight, and then inoculated into the fermentation medium at a concentration of 0.02 at the initial OD600, cultured at 28 ° C, 180 rpm for 2 to 3 days, and the bacteria were measured at intervals of time. Liquid OD600.
  • Figure 2 shows the growth curves of Pseudomonas aeruginosa HT66 and its different genetically engineered strains. It can be seen from the figure that knocking out the parS gene has no effect on the growth of the strain; knocking out the lon gene will slightly decrease the OD600 value of the strain during the stationary phase, and the OD600 value of the double knockout strain HT66 ⁇ lon ⁇ parS is the lowest, indicating that the two gene pairs The growth of the bacteria has Complex effects.
  • the activated P. fluorescens HT66 and its genetically engineered strains HT66 ⁇ parS, HT66 ⁇ lon and HT66 ⁇ lon ⁇ parS were inoculated into KB medium and placed in a 28 ° C constant temperature shaker (180 rpm) until the OD600 was 0.5-2.0. During the interval, the above bacterial solution is added to a fresh KB fermentation medium at a volume ratio of 1 to 10:100, shake culture at 24 to 30 ° C, the shaking speed of the shaker is 100 to 300 rpm, and the culture is carried out for 24 to 72 hours. Bacteria.
  • 0.5 mL of the fermentation broth was added to 3 mL of ethyl acetate for extraction, and 0.2 mL of the organic phase was dried and dissolved in acetonitrile, and the yield of phenazine-1-carboxamide in the fermentation broth was measured by HPLC.
  • the mobile phase was acetonitrile-25 mM ammonium acetate
  • the column was WondaSilC18-WRreverse phasecolumn (5 ⁇ m; 4.6 ⁇ 250 mm, Shimadzu, Japan)
  • the detection wavelength was 254 nm
  • the detection conditions were: 0 to 2 min, 8% acetonitrile-92% 25 mM ammonium acetate, 2- At 20 min, the acetonitrile concentration was increased from 8% to 60%, and the ammonium acetate concentration was decreased from 92% to 40%, 20 to 21 minutes, and 8% acetonitrile to 92% 25 mM ammonium acetate.
  • Figure 3 is a graph showing the changes in yield of phenazine-1-carboxamide in different genetically engineered strains of Pseudomonas aeruginosa HT66 and wild strains.
  • knocking out the parS gene caused the PCN yield to increase from 425 mg/L to 1102 mg/L in the wild strain, and knocking out the lon gene increased the yield of PCN from 425 mg/L to 2053 mg/L in the wild strain;
  • the PCN yield of the double mutant strain reached 2425 mg/L, which was 5.71 times that of the wild strain.
  • This example provides a genetically engineered strain with high yield of phenazine-1-carboxamide, specifically by knocking out Pseudomonas chlororaphis HT66 CCTCC NO: M 2013467 rpeA, psrA single mutant or double mutation.
  • Gene engineering strains are obtained from one or two genes of the lon gene and the parS gene of the strain genome;
  • one of the lon gene and the parS gene of the genetically engineered strains HT66 ⁇ rpeA, HT66 ⁇ psrA, HT66 ⁇ rpeA ⁇ psrA was knocked out or two simultaneous knockouts were obtained, including genetically engineered strains HT66 ⁇ lon ⁇ rpeA, HT66 ⁇ parS ⁇ rpeA, HT66 ⁇ lon ⁇ parS ⁇ rpeA.
  • Figure 4 is a growth curve of HT66 ⁇ lon ⁇ rpeA genetically engineered bacteria
  • Figure 5 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ lon ⁇ rpeA genetically engineered bacteria
  • Figure 6 is a growth curve of HT66 ⁇ parS ⁇ rpeA genetically engineered bacteria
  • Figure 7 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ parS ⁇ rpeA genetically engineered bacteria
  • Figure 8 is a growth curve of HT66 ⁇ lon ⁇ parS ⁇ rpeA genetically engineered bacteria
  • Figure 9 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ lon ⁇ parS ⁇ rpeA genetically engineered bacteria
  • Figure 10 is a growth curve of HT66 ⁇ lon ⁇ psrA genetically engineered bacteria
  • Figure 11 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ lon ⁇ psrA genetically engineered bacteria
  • Figure 12 is a growth curve of HT66 ⁇ psrA ⁇ parS genetically engineered bacteria
  • Figure 13 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ psrA ⁇ parS genetically engineered bacteria
  • Figure 14 is a growth curve of HT66 ⁇ lon ⁇ parS ⁇ psrA genetically engineered bacteria
  • Figure 15 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ lon ⁇ parS ⁇ psrA genetically engineered bacteria
  • Figure 16 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ lon ⁇ parS ⁇ psrA ⁇ rpeA genetically engineered bacteria
  • Figure 17 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ lon ⁇ psrA ⁇ rpeA genetically engineered bacteria
  • Figure 18 is a graph showing the yield of phenazine-1-carboxamide of HT66 ⁇ parS ⁇ psrA ⁇ rpeA genetically engineered bacteria.
  • the present embodiment relates to a method for preparing an HT66 ⁇ pykA genetically engineered strain by using an mutated mutation method using the HT66 genome as a template, comprising the following steps:
  • primers were designed and synthesized using primer 5 as a reference, and the primer sequences were as follows, and the restriction enzyme sites of restriction enzymes Hind III and Sac I were underlined.
  • pykA1 CC AAGCTT ATGTCCGTCCGTCGTACCAA (SEQ ID NO. 13)
  • pykA2 AC GAGCTC TCAGACCATTGGGTCGCCA (SEQ ID NO. 14)
  • primers for the Kana resistance gene were designed and synthesized.
  • the primer sequences were as follows, and the restriction endonuclease site of restriction endonuclease Xho I was underlined.
  • pBb-Kan-F CC CTCGAG GGAATTGCCAGCTGGGGCGC SEQ ID NO.
  • pBb-Kan-R CC CTCGAG TCAGAAGAACTCGTCAAGAAG SEQ ID NO.16
  • pykA gene (1452bp) and Kan gene (1000bp) were amplified by PCR using pykA1/2 and pBb-Kan-F/R primers. After purification of the amplified product, the Kan gene PCR product was first. Store in a -20° refrigerator. Use pykA gene and pEX18Tc plasmid to utilize Hind III Seco-digestion was performed with Sac I, and the enzyme was digested with restriction enzyme digestion. The E. coli DH5a competent cells were ligated and transformed at 16 ° C, and positive clones were obtained by Tc (20 ppm) resistant plate screening.
  • the pEX-pykA plasmid was extracted, and Xho I was digested with the Kan gene. After tapping recovery, the cells were ligated overnight at 16 ° C and transformed into DH5a. The positive clones were obtained by screening recombinants with Tc (20 ppm) and Kan (50 ppm) resistant plates. The obtained plasmid pEX-pykA-Kan was extracted and transferred into E. coli SM10 competent cells.
  • SM10 transfected into the pEX-pykA-Kan plasmid was subjected to amphiphilic hybridization with strain HT66, and subjected to single exchange screening by Tc (150 ppm), Kan (50 ppm), and Sp (100 ppm) LB-resistant plates, containing 15% sucrose and Kan ( 50ppm), Sp (100ppm) LB plate was subjected to double exchange screening, external primer (pykA1/2) PCR verification, genome amplification amplification and sequencing, and finally the insertion mutant HT66 ⁇ pykA was obtained.
  • the inserted mutant strain can amplify a fragment of about 2500 bp in length (the sum of the size of the pykA and kan genes), while the unbroken wild strain can amplify a fragment of about 1500 bp (pykA).
  • the size is 1452 bp), and the results are shown in FIG.
  • M is Marker
  • L1 is a blank control
  • L2 using the genome of strain HT66 as a template
  • L3 using the genome of strain HT66 ⁇ pykA as a template.
  • strains HT66 and HT66 ⁇ pykA were determined by liquid culture as shown in FIG. The results showed that knocking out the pykA gene in HT66 did not affect its growth, indicating that the genetically engineered bacteria had good stability and was beneficial for further modification of the strain.
  • the present embodiment relates to the preparation of a genetically engineered strain of high-yield PCN, which is a strain of high-yield PCN obtained by multiple mutations of Pseudomonas aeruginosa HT66. Specifically, the following steps are included:
  • the primers were designed and knocked out using primer 5, and the primer sequences are as follows (underlined for XbaI and HindIII cleavage sites):
  • pykAF1 CCGGGGATCC TCTAGA AAGATCGTTACAACGCGGTCG (SEQ ID NO. 17)
  • pykAR1 TGGTACGACGGACGGACATG (SEQ ID NO. 18)
  • pykAR2 GGCCAGTGCC AAGCTT CGAGTTCGGTTCCAGCCTG (SEQ ID NO. 20)
  • the P3 genome was used as a template to amplify the upstream and downstream homologous arm fragments of pykA by PCR using pykAF1, pykAR1 and pykAF2, pykAR2.
  • the upstream and downstream homology arms were connected to the in-Fusion HD Cloning Kit method.
  • the pk18mobsacB plasmid was digested and transformed into E. coli DH5a, and screened by blue-white spot.
  • the leukoplakia was subjected to PCR verification using primers pykAF1 and pykAR2 and then sent to sequencing.
  • the correctly sequenced plasmid pK18-pykA was transformed into E. coli S17, and combined with strain P3 for transfer, single-exchange screening by Kan and Amp-resistant plates, 15% (w/v) sucrose plate double exchange screening, external primer PCR verification The genome was amplified and sequenced. Finally, the pykA knockout mutant of strain P3 was obtained and named as P3 ⁇ pykA. PCR amplification was performed using pykAF1 and pykAR2 primers. The knockout strain amplified a homologous arm fragment of pykA upstream and downstream of about 1100 bp.
  • L1 is a blank control
  • L2 using the genome of strain P3 as a template
  • L3 using the genome of mutant P3 ⁇ pykA as a template.
  • the growth curves of the strains P3 and P3 ⁇ pykA were simultaneously determined by liquid culture in KB medium, as shown in FIG. The results showed that knocking out the pykA gene in strain P3 did not affect its growth, indicating that the genetically engineered strain has very good stability and is beneficial to its industrial application.
  • the present embodiment relates to a method for synthesizing PCN using the genetically engineered strain prepared in Example 7, comprising the following steps:
  • strains HT66 ⁇ pykA and HT66wt wild strain HT66 as control were respectively activated twice on KB solid plate, then cultured in 5 mL KB liquid medium, cultured to log phase, and transferred to 2 mL in a ratio of 2%.
  • the flask of KB medium was fermented. The fermentation conditions were 28 ° C, 180 rpm. Samples were taken periodically and the strain PCN yield was determined by HPLC.
  • the mobile phase was acetonitrile-25 mM ammonium acetate
  • the column was WondaSilC18-WRreverse phasecolumn (5 ⁇ m; 4.6 ⁇ 250 mm, Shimadzu, Japan)
  • detection wavelength was 254 nm
  • detection conditions 0-2 min, 8% acetonitrile-92% 25 mM ammonium acetate
  • the PCN yield of strain HT66 and strain HT66 ⁇ pykA was measured, and the results are shown in FIG. From the map See, knocking out the pykA gene can significantly increase PCN yield.
  • the PCN yield of the control strain HT66 is 552 mg/L, while the PCN yield of the mutant strain is 1038 mg/L at 48 h, an increase of 88%. It can be seen that knocking out the pykA gene in Pseudomonas aeruginosa HT66 can effectively change the flow of substances and facilitate the accumulation of PCN.
  • the present embodiment relates to a method for producing PCN using the genetically engineered strain prepared in Example 8, comprising the following steps:
  • the strains P3 ⁇ pykA and P3 were first activated on KB solid plates, respectively, and then incubated in 5 mL KB liquid medium. After they were grown to log phase, they were inoculated separately into a flask containing 60 mL of KB medium. Fermentation. The initial inoculum size OD600 was 0.02. The fermentation conditions were 28 ° C, 180 rpm. Timed sampling was performed to determine the PCN yield in the fermentation broth of the strains P3 ⁇ pykA and P3, respectively, and the results are shown in FIG.
  • the invention specifically uses Pseudomonas sphaeroides HT66 as a starting strain, and knocks out the lon and parS negative regulatory genes in the phenazine synthesis process from the HT66 strain and its known mutant strain genome by genetic engineering technology, respectively.
  • Lon, parS gene single gene mutant or double genetic engineering strain the yield of phenazine-1-carboxamide (CAS No. 550-89-0, PCN for short) is greatly improved, which can be more effective due to Biological control.

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Abstract

一种吩嗪-1-甲酰胺(PCN)的基因工程菌株及其制备方法和用途；所述基因工程菌株具体是敲除绿针假单胞菌(Pseudomonas chlororaphis) HT66 CCTCC NO: M 2013467及其衍生物基因组的lon基因、parS基因中的一个或两个基因而得到基因工程菌株；或者，敲除绿针假单胞菌HT66 CCTCC NO:M 2013467 rpeA、psrA单突变株或者双突变株基因组的lon基因、parS基因中的一个或两个基因而得到基因工程菌株，或者敲除绿针假单胞菌HT66 CCTCC NO:M 2013467及其衍生物中的pykA基因；其中，所述单突变株为HT66ΔrpeA、HT66ΔpsrA，双突变株为HT66ΔrpeAΔpsrA。将所述绿针假单胞菌基因组中lon和parS基因删除后，吩嗪-1-甲酰胺的发酵产量最高可达3594mg/L，可以用于吩嗪-1-甲酰胺的工业化生产与农业应用。

Description

高产吩嗪-1-甲酰胺的基因工程菌株及其构建方法和用途 技术领域
本发明属于基因工程领域，涉及假单胞菌基因工程菌株及其构建方法和用途，尤其是一株高产吩嗪-1-甲酰胺的基因工程菌株及其构建方法和用途。
背景技术
我国常见的农作物病害生物达1600种，每年造成的损失约为1000亿人民币，严重地影响了我国农业的发展。近些年，化学农药的大规模使用导致了病虫害的抗药性不断增强。此外，高浓度和毒性的化学农药的应用，对农田、水环境也造成了很大的污染，严重威胁着我国的粮食安全和生命健康。因此，低毒性、低残留和不易产生抗药性的生物农药获得了各国政府和农药企业的极大关注。
与化学农药相比，生物农药具有毒性低、对环境友好和不易产生抗药性等明显优势，已经吸引了越来越多学者的关注并逐渐应用到生物防治实践当中。其中，上海交通大学彭华松课题组率先利用铜绿假单胞菌株M18开发出了具有广谱、高效、安全和能有效控制真菌性根腐和茎腐的生物农药吩嗪-1-羧酸(phenazine-1-carboxylic acid，简称PCA，CAS号为2538-68-3)，定名为“申嗪霉素”，主要用于防治水稻纹枯病、小麦赤霉病、黄瓜和西瓜的枯萎病、甜瓜蔓枯病、辣椒根腐病等，并获得了农业部颁发的新农药药证。
但最近相关研究发现，与PCA相比，另一种吩嗪类抗生素吩嗪-1-甲酰胺(phenazine-1-carboxamide，PCN)具有更好的安全性、稳定性及对植物病原菌的抑菌活性，在防治水稻纹枯病和小麦赤霉病等方面具有重要的应用价值。据报道，目前能合成PCN的微生物菌株有绿针假单胞菌PCL1391，铜绿假单胞菌PAO1，PUPa3，PUP6，MML221等，但由于PCN产率较低，尚未能大规模推广应用。上海交通大学彭华松课题组对筛选的一株绿针假单胞菌HT66进行基因工程改造后，PCN的产量已经达到1800mg/L并申报了中国发明专利(CN201310566864.5)，但是该产量距离PCN的工业化应用还存在一定的距离。因此，通过基因工程技术进一步改造该菌株将有利于提高PCN产量，实现其农业应用。
发明内容
针对现有技术中的缺陷，本发明的目的是提供一株高产吩嗪-1-甲酰胺的基因工程菌株及其构建方法，克服现有菌株生产吩嗪-1-甲酰胺产量和效率较低的缺陷，提供一种用于生产吩嗪-1-甲酰胺的基因工程菌株及其用途。
本发明的目的是通过以下技术方案来实现的：
第一方面，本发明涉及、一株高产吩嗪-1-甲酰胺的基因工程菌株，其具体是敲除绿针假单胞菌(Pseudomonas chlororaphis)HT66 CCTCC NO:M 2013467及其衍生物基因组的lon基因、parS基因中的一个或两个基因而得到基因工程菌株；
或者，敲除绿针假单胞菌(Pseudomonas chlororaphis)HT66 CCTCC NO:M 2013467rpeA、psrA单突变株或者双突变株基因组的lon基因、parS基因中的一个或两个基因而得到基因工程菌株；其中，所述单突变株为HT66ΔrpeA、HT66ΔpsrA，双突变株为HT66ΔrpeAΔpsrA；
或者敲除了绿针假单胞菌(Psedunomonaschlororaphis)HT66 CCTCC NO:M2013467及其衍生物基因组中的pykA基因，即得。
作为优选方案，所述lon基因的碱基序列如SEQ ID NO.1所示；所述parS基因的碱基序列如SEQ ID NO.2所示，所述pykA基因的碱基序列如SEQ ID NO.11所示，所述pykA基因的对应蛋白质的氨基酸序列如SEQ ID NO.12所示。
作为优选方案，所述基因工程菌株包括HT66Δlon、HT66ΔparS、HT66ΔlonΔparS、HT66ΔlonΔrpeA、HT66ΔparSΔrpeA、HT66ΔlonΔpsrA、HT66ΔpsrAΔparS、HT66ΔlonΔparSΔrpeA、HT66ΔlonΔparSΔpsrA、HT66ΔlonΔpsrAΔrpeA、HT66ΔparSΔpsrAΔrpeA、HT66ΔlonΔparSΔpsrAΔrpeA、HT66ΔpykA。
第二方面，本发明提供了一种根据前述高产吩嗪-1-甲酰胺的基因工程菌株的构建方法，其具体是：敲除绿针假单胞菌(Pseudomonas chlororaphis)HT66 CCTCC NO:M2013467及其衍生物基因组的lon基因、parS基因中的一个基因或两个基因，即可；
或者，敲除绿针假单胞菌(Pseudomonas chlororaphis)HT66 CCTCC NO:M 2013467rpeA、psrA单突变株或者双突变株基因组的lon基因、parS基因中的一个或两个基因而得到基因工程菌株；其中，所述单突变株为HT66ΔrpeA、HT66ΔpsrA，双突变株为HT66ΔrpeAΔpsrA；
或者，敲除了绿针假单胞菌(Psedunomonaschlororaphis)HT66 CCTCC NO:M2013467及其衍生物基因组中的pykA基因，即得。
作为优选方案，所述构建方法包括：
i、扩增lon基因或parS基因片段的上下游同源臂；
ii、采用融合PCR法连接上下游同源臂并插入pK18mobsacB质粒中得lon基因或parS基因重组质粒；
iii、(a)使lon基因重组质粒上的lon基因上下游同源臂片段与HT66、HT66ΔparS、HT66ΔrpeA、HT66ΔrpeAΔparS、HT66ΔpsrA、HT66ΔpsrAΔparS、HT66ΔrpeAΔpsrA、HT66ΔrpeAΔpsrAΔparS基因组中的lon基因发生同源重组；
或(b)使parS基因重组质粒上的parS基因上下游同源臂片段与HT66、HT66Δlon、HT66ΔrpeA、HT66ΔrpeAΔlon、HT66ΔpsrA、HT66ΔpsrAΔlon、HT66ΔrpeAΔpsrA、HT66ΔrpeAΔpsrAΔlon基因组中的parS基因发生同源重组；
筛选阳性克隆，即得；
或，敲除所述pykA基因的方法为插入突变法或无痕敲除法；
所述插入突变法敲除pykA基因包括如下步骤：
S1、扩增pykA基因片段，并插入pEX18Tc质粒中；
S2、扩增卡那霉素或庆大霉素等抗性基因，插入pykA基因中间，使pykA基因发生插入突变；
S3、将突变后的pykA基因与HT66基因组中的pykA基因发生同源重组，根据抗性筛选出阳性克隆，即可；
所述无痕敲除pykA基因包括如下步骤：
A1、扩增pykA基因的上下游同源臂；
A2、融合PCR法连接上下游同源臂并插入pK18mobsacB质粒中；
A3、使重组质粒上的pykA基因的上下游同源臂片段与HT66基因组发生同源重组，利用蔗糖压力和抗性筛选出阳性克隆，即可。
第三方面，本发明提供了一种基于前述基因工程菌株的吩嗪-1-甲酰胺合成方法，其具体包括如下步骤：将所述基因工程菌株的种子发酵液接种于KB发酵培养基中培养，即可。
作为优选方案，所述种子发酵液的制备具体为：将所述基因工程菌株接种于KB培养基中，置于28℃、180转/分下培养至OD600为0.5～2.0，即得。
作为优选方案，所述培养的条件具体为：26～34℃、100～300转/分钟、24～72h。
作为优选方案，所述种子发酵液与所述KB发酵培养基的体积比为(1～10):100。
第四方面，本发明提供了一种基于前述基因工程菌株的吩嗪-1-甲酰胺合成方法，其 具体包括如下步骤：将所述基因工程菌株接种于KB液体培养基中培养，即可。
第五方面，本发明提供了一种用于前述的基因工程菌株的培养基，其包括按重量份数计的如下组分：
Figure PCTCN2017098480-appb-000001
第六方面，本发明提供了一种如前述的高产吩嗪-1-甲酰胺的基因工程菌株在生产吩嗪-1-甲酰胺中的用途。
作为优选方案，所述基因工程菌株在制备吩嗪-1-甲酰胺时的条件为：好氧培养；温度：26～34℃；pH：6～8；转速100～350rpm。
与现有技术相比，本发明的具备如下有益效果：
本发明具体是以绿针假单胞菌HT66为出发菌株，通过基因工程技术从HT66菌株基因组中分别或者同时敲除吩嗪合成过程中的lon、parS负调控基因，构建HT66Δlon、HT66ΔparS、HT66ΔlonΔparS、HT66ΔpykA基因基因工程菌株，使得吩嗪-1-甲酰胺(phenazine-1-carboxamide,CAS号为550-89-0，简称PCN)的产量大幅提高，能够更加有效由于生物防治。其中，基因工程菌株HT66ΔlonΔparS具有最佳效果。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为绿针假单胞菌HT66和lon敲除突变株中分别以内外部引物PCR扩增lon基因及其上下游片段的电泳图；
其中，从左到右依次为marker，HT66Δlon，HT66，空白对照；
图2为HT66Δlon、HT66ΔparS、HT66ΔlonΔparS基因工程菌的生长曲线图；
图3为HT66HT66Δlon、HT66ΔparS、HT66ΔlonΔparS基因工程菌的吩嗪-1-甲酰胺产量图；
图4为HT66ΔlonΔrpeA基因工程菌的生长曲线图；
图5为HT66ΔlonΔrpeA基因工程菌的吩嗪-1-甲酰胺产量图；
图6为HT66ΔparSΔrpeA基因工程菌的生长曲线图；
图7为HT66ΔparSΔrpeA基因工程菌的吩嗪-1-甲酰胺产量图；
图8为HT66ΔlonΔparSΔrpeA基因工程菌的生长曲线图；
图9为HT66ΔlonΔparSΔrpeA基因工程菌的吩嗪-1-甲酰胺产量图；
图10为HT66ΔlonΔpsrA基因工程菌的生长曲线图；
图11为HT66ΔlonΔpsrA基因工程菌的吩嗪-1-甲酰胺产量图；
图12为HT66ΔpsrAΔparS基因工程菌的生长曲线图；
图13为HT66ΔpsrAΔparS基因工程菌的吩嗪-1-甲酰胺产量图；
图14为HT66ΔlonΔparSΔpsrA基因工程菌的生长曲线图；
图15为HT66ΔlonΔparSΔpsrA基因工程菌的吩嗪-1-甲酰胺产量图；
图16为HT66ΔlonΔparSΔpsrAΔrpeA基因工程菌的吩嗪-1-甲酰胺产量图；
图17为HT66ΔlonΔpsrAΔrpeA基因工程菌的吩嗪-1-甲酰胺产量图；
图18为HT66ΔparSΔpsrAΔrpeA基因工程菌的吩嗪-1-甲酰胺产量图；
图19为不同菌株中pykA基因扩增产物的电泳图(L1为空白对照，L2:以野生株HT66的基因组为模板，L3:以突变株HT66ΔpykA的基因组为模板)；
图20为野生株HT66与突变菌株HT66ΔpykA的生长曲线对比图；
图21为不同菌株中pykA基因扩增产物的电泳图(L1为空白对照，L2:以菌株P3的基因组为模板，L3:以突变株P3ΔpykA的基因组为模板)
图22为菌株P3和突变株P3ΔpykA的生长曲线对比图；
图23为野生株HT66和突变株HT66ΔpykA的PCN发酵曲线；
图24为菌株P3和突变菌株P3ΔpykA的PCN发酵曲线。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这些都属于本发明的保护范围。
本发明的绿针假单胞菌(Pseudomonas choloraphis)HT66，该菌株已在中国典型培养物保藏中心(简称CCTCC)保藏，保藏单位地址为：中国.武汉.武汉大学，邮编为：430072，保藏日期为：2013年10月12日，保藏编号为：CCTCC NO:M 2013467。
实施例1、lon基因体外突变体的构建
根据绿针假单胞菌HT66基因组中lon基因及其上下游序列设计引物(见表1)：
表1
Figure PCTCN2017098480-appb-000002
以该基因组DNA为模板，扩增基因组中的相应片段。
将上下游PCR产物通过融合PCR连接，并将融合PCR产物与pK18mobsacB载体分别使用EcoRI和XbaI酶切，过柱回收，使用T4连接酶连接，获得体外突变质粒。
用体外突变质粒转化大肠杆菌S17。将携带重组质粒的S17菌株充分活化，接种于含有卡那霉素50mg/L的LB培养基中，37℃，180转/分培养12h。将充分活化的HT66菌株接种于LB培养基中，28℃，180转/分培养12h。5000转/分收集并洗涤两种菌体，LB培养基重悬并以HT66：S17浓度比为3:1的比例混合与EP管中，静置1h；取混合菌液点在LB平板中，28℃培养24h；刮取长出来的混合菌落于LB中重悬，稀释涂布于卡那霉素50mg/L，氨苄霉素100mg/L的LB平板上，28℃培养2～3d后挑取单克隆涂布10％蔗糖平板中，28℃培养1～2d；挑取蔗糖平板上的单克隆，分别点在卡那霉素50mg/L的抗性LB平板及无抗LB平板上，培养12～24h；挑取在卡那霉素50mg/L抗性平板上不生长，但在无抗平板上能够正常生长的单菌落，即为同源重组成功的菌落。PCR验证HT66菌株的lon基因被敲除，并命名为HT66Δlon。
图1为绿针假单胞菌HT66和lon敲除突变株中分别以内外部引物PCR扩增lon基因及其上下游片段的电泳图；其中，M，Marker；Δ，lon基因敲除株HT66Δlon；WT，野生株HT66；N，空白对照组。左侧引物为内部引物lon-F/lon-R，右侧引物为外部引物lon-F1/lon-R2。内外部引物扩增结果表明，lon基因已被成功敲除。
实施例2、parS基因体外突变体的构建
根据绿针假单胞菌HT66基因组中parS及上下游序列设计引物(见表2)：
表2
Figure PCTCN2017098480-appb-000003
以该基因组DNA为模板，扩增基因组中的相应片段。
将上下游PCR产物通过融合PCR连接，并将融合PCR产物与pK18mobsacB载体分别使用BamHI和HindIII酶切，过柱回收，使用T4连接酶连接，获得体外突变质粒。
用所得体外突变质粒转化大肠杆菌S17。将携带重组质粒的S17菌株充分活化，接种于含有卡那霉素50mg/L的LB培养基中，37℃，180转/分培养12h。将充分活化的HT66菌株接种于LB培养基中，28℃，180转/分培养12h。5000转/分收集并洗涤两种菌体，LB培养基重悬并以HT66：S17浓度比为3:1的比例混合与EP管中，静置1h；取混合菌液点在LB平板中，28℃培养24h；刮取长出来的混合菌落于LB中重悬，稀释涂布于卡那霉素50mg/L，氨苄霉素100mg/L的LB平板上，28℃培养2～3d后挑取单克隆涂布10％蔗糖平板中，28℃培养1～2d；挑取蔗糖平板上的单克隆，分别点在卡那霉素50mg/L的抗性LB平板及无抗LB平板上，培养12～24h；挑取在卡那霉素50mg/L抗性平板上不生长，但在无抗平板上能够正常生长的单菌落，即为同源重组成功的菌落。PCR验证HT66菌株的parS基因被敲除，并命名为HT66ΔparS。
实施例3、lon和parS双突变株HT66ΔlonΔparS的构建
将携带parS重组质粒的S17菌株充分活化，接种于含有卡那霉素50mg/L的LB培养基中，37℃，180转/分培养12h。将充分活化的HT66Δlon菌株接种于LB培养基中，28℃，180转/分培养12h。5000转/分收集并洗涤两种菌体，LB培养基重悬并以HT66Δlon：S17浓度比为3:1的比例混合与EP管中，静置1h；取混合菌液点在LB平板中，28℃培养24h；刮取长出来的混合菌落于LB中重悬，稀释涂布于卡那霉素50mg/L，氨苄霉素100mg/L的LB平板上，28℃培养2～3d后挑取单克隆涂布10％蔗糖平板中，28℃培养1～2d；挑取蔗糖平板上的单克隆，分别点在卡那霉素50mg/L的抗性LB平板及无抗LB平板上，培养12～24h；挑取在卡那霉素50mg/L抗性平板上不生长，但在无抗平板上能够正常生长的单菌落，即为同源重组成功的菌落。PCR验证HT66Δlon菌株的parS基因被敲除，并命名为HT66ΔlonΔparS。
实施例4、假单胞菌HT66及基因工程菌株HT66ΔlonΔparS的生长曲线测定
将菌株充分活化并接种于新鲜的KB培养基中，培养过夜后以最初OD600为0.02的浓度接种于发酵培养基，28℃，180转/分培养2～3d，并以一定的时间间隔测定菌液OD600。
图2为绿针假单胞菌HT66及其不同基因工程菌株的生长曲线。由图可知，敲除parS基因对菌株的生长影响不打；敲除lon基因会使菌株稳定期的OD600值略微下降，其中以双敲除株HT66ΔlonΔparS的稳定期OD600值最低，说明两个基因对菌体的生长有着 复杂的影响。
实施例5、基因工程菌HT66ΔlonΔparS中PCN的生物合成
将活化后的绿针假单胞菌HT66及其基因工程菌株HT66ΔparS、HT66Δlon、HT66ΔlonΔparS分别接种于KB培养基中，置于28℃恒温摇床(180转/分)培养至OD600为0.5～2.0之间时，将上述菌液以1～10:100的体积比，加入到新鲜的KB发酵培养基中，24～30℃振荡培养，摇床转速100～300转/分钟，培养24～72h后收取菌液。取发酵液0.5mL加入3mL乙酸乙酯充分萃取后，取0.2mL有机相吹干后用乙腈溶解，并通过HPLC检测发酵液中吩嗪-1-甲酰胺产量。
HPLC检测条件：
流动相为乙腈-25mM乙酸铵，色谱柱为WondaSilC18-WRreversephasecolumn(5μm；4.6X250mm,Shimadzu,Japan)，检测波长为254nm，检测条件：0～2min，8％乙腈-92％25mM乙酸铵，2-20min，乙腈浓度从8％升至60％，乙酸铵浓度从92％下降至40％，20～21min，8％乙腈-92％25mM乙酸铵。
图3为绿针假单胞菌HT66不同基因工程菌株及野生株中吩嗪-1-甲酰胺的产量变化图。由图可知，敲除parS基因导致了PCN的产量由野生株的425mg/L上升到1102mg/L，敲除lon基因使PCN的产量由野生株的425mg/L上升到2053mg/L；两个基因的双突变株中PCN产量达到2425mg/L，为野生株的5.71倍。
实施例6
本实施例提供了一株高产吩嗪-1-甲酰胺的基因工程菌株，具体是通过敲除绿针假单胞菌(Pseudomonas chlororaphis)HT66 CCTCC NO:M 2013467 rpeA、psrA单突变株或者双突变株基因组的lon基因、parS基因中的一个或两个基因而得到基因工程菌株；
其中，绿针假单胞菌(Pseudomonas chlororaphis)HT66 CCTCC NO:M 2013467rpeA、psrA单突变株分为HT66ΔrpeA、HT66ΔpsrA，双突变株为HT66ΔrpeAΔpsrA；上述单突变株及双突变株相关信息公开于在先申请CN 103642714A中。
参照实施例1至3提供的方法，将基因工程菌株HT66ΔrpeA、HT66ΔpsrA、HT66ΔrpeAΔpsrA的lon基因、parS基因中的一个进行敲除或者两个同时敲除，即得；包括基因工程菌株HT66ΔlonΔrpeA、HT66ΔparSΔrpeA、HT66ΔlonΔparSΔrpeA、HT66ΔlonΔpsrA、HT66ΔpsrAΔparS、HT66ΔlonΔparSΔpsrA、HT66ΔlonΔparSΔpsrAΔrpeA、HT66ΔlonΔpsrAΔrpeA、HT66ΔparSΔpsrAΔrpeA。上述基因工程菌株生长情况及PCN产量情况如图4至图18所示。其中：
图4为HT66ΔlonΔrpeA基因工程菌的生长曲线图；
图5为HT66ΔlonΔrpeA基因工程菌的吩嗪-1-甲酰胺产量图；
图6为HT66ΔparSΔrpeA基因工程菌的生长曲线图；
图7为HT66ΔparSΔrpeA基因工程菌的吩嗪-1-甲酰胺产量图；
图8为HT66ΔlonΔparSΔrpeA基因工程菌的生长曲线图；
图9为HT66ΔlonΔparSΔrpeA基因工程菌的吩嗪-1-甲酰胺产量图；
图10为HT66ΔlonΔpsrA基因工程菌的生长曲线图；
图11为HT66ΔlonΔpsrA基因工程菌的吩嗪-1-甲酰胺产量图；
图12为HT66ΔpsrAΔparS基因工程菌的生长曲线图；
图13为HT66ΔpsrAΔparS基因工程菌的吩嗪-1-甲酰胺产量图；
图14为HT66ΔlonΔparSΔpsrA基因工程菌的生长曲线图；
图15为HT66ΔlonΔparSΔpsrA基因工程菌的吩嗪-1-甲酰胺产量图；
图16为HT66ΔlonΔparSΔpsrAΔrpeA基因工程菌的吩嗪-1-甲酰胺产量图；
图17为HT66ΔlonΔpsrAΔrpeA基因工程菌的吩嗪-1-甲酰胺产量图；
图18为HT66ΔparSΔpsrAΔrpeA基因工程菌的吩嗪-1-甲酰胺产量图。
实施例7
本实施例涉及一种以HT66基因组为模板，利用插入突变法制备HT66ΔpykA基因工程菌株的方法，包括如下步骤：
1.引物设计
以pykA基因序列为参考，利用primer 5设计引物并合成，引物序列如下，下划线表示限制性内切酶Hind III和Sac I的酶切位点。
pykA1:CCAAGCTTATGTCCGTCCGTCGTACCAA(SEQ ID NO.13)
pykA2:ACGAGCTCTCAGACCATTGGGTCGCCA(SEQ ID NO.14)
以质粒pBbB5k为模板，设计卡那抗性基因(Kan)的引物并合成，引物序列如下，下划线表示限制性内切酶Xho I的酶切位点。
pBb-Kan-F:CCCTCGAGGGAATTGCCAGCTGGGGCGC SEQ ID NO.15
pBb-Kan-R:CCCTCGAGTCAGAAGAACTCGTCAAGAAG SEQ ID NO.16
2.插入突变重组质粒构建
以HT66基因组和pBbB5k质粒为模板，利用pykA1/2和pBb-Kan-F/R引物通过PCR分别扩增pykA基因(1452bp)和Kan基因(1000bp)，扩增产物纯化后，Kan基因PCR产物先置于-20°冰箱保存。将pykA基因和pEX18Tc质粒利用Hind III 和Sac I分别进行双酶切，酶切体系切胶回收，16℃连接并转化E.coli DH5a感受态细胞，通过Tc(20ppm)抗性平板筛选获得阳性克隆。提取pEX-pykA质粒，与Kan基因一起分别进行Xho I单酶切，割胶回收后16℃连接过夜并转化DH5a，使用含Tc(20ppm)，Kan(50ppm)的抗性平板筛选重组子获得阳性克隆，提取获得的质粒pEX-pykA-Kan，转入E.coli SM10感受态细胞。
3.插入突变菌株筛选
将转入pEX-pykA-Kan质粒的SM10与菌株HT66进行双亲杂交，通过Tc(150ppm)，Kan(50ppm)，Sp(100ppm)的LB抗性平板进行单交换筛选，含15％蔗糖和Kan(50ppm)，Sp(100ppm)的LB平板进行双交换筛选，外部引物(pykA1/2)PCR验证，提基因组扩增送测序，最后获得插入突变株HT66ΔpykA。
利用pykA1和pykA2引物进行PCR验证，插入突变菌株可扩增出长约2500bp的片段(pykA和kan基因大小之和)，而未敲除的野生菌株则可扩增出长约1500bp的片段(pykA大小为1452bp)，结果如图19所示。图19中：M为Marker，L1为空白对照，L2:以菌株HT66的基因组为模板，L3:以菌株HT66ΔpykA的基因组为模板。
通过液体培养，测定菌株HT66和HT66ΔpykA生长曲线如图20所示。结果显示在HT66中敲除pykA基因不会影响其生长，表明该基因工程菌的稳定性好，有利于对该菌进行进一步改造。
实施例8
本实施例涉及一种高产PCN的基因工程菌株的制备，出发菌株P3是绿针假单胞菌HT66经过多重突变后获得的高产PCN的菌株。具体包括如下步骤：
1、引物设计：
以pykA基因上下游序列为参考，利用primer 5设计敲除引物并合成，引物序列如下(下划线表示XbaI和HindIII酶切位点)：
pykAF1:CCGGGGATCCTCTAGAAAGATCGTTACAACGCGGTCG(SEQ ID NO.17)
pykAR1:TGGTACGACGGACGGACATG(SEQ ID NO.18)
pykAF2:TGTCCGTCCGTCGTACCAACCCAATGGTCTGAGCCACC(SEQ ID NO.19)
pykAR2:GGCCAGTGCCAAGCTTCGAGTTCGGTTCCAGCCTG(SEQ ID NO.20)
2、重组质粒构建：
以P3基因组为模板，利用pykAF1、pykAR1和pykAF2、pykAR2通过PCR扩增pykA上下游同源臂片段，PCR产物纯化后，利用In-Fusion HD Cloning Kit方法将该上下游同源臂连接至已进行酶切过的pk18mobsacB质粒上并转化E.coli DH5a，通过蓝白斑筛选，白斑菌落利用引物pykAF1和pykAR2进行PCR验证后送测序。
3、突变菌株筛选：
将测序正确的质粒pK18-pykA转化至E.coli S17，与菌株P3进行结合转移，通过Kan和Amp抗性平板单交换筛选，15％(w/v)蔗糖板双交换筛选，外部引物PCR验证，提基因组扩增送测序，最后获得菌株P3的pykA敲除突变株，命名为P3ΔpykA，利用pykAF1和pykAR2引物PCR扩增，敲除菌株可扩增出长约1100bp的pykA上下游同源臂片段，而未敲除的野生菌株则可扩增出长约2500bp的片段(pykA大小为1452bp)，表明pykA基因成功敲除，结果如图21所示。图21中，L1为空白对照，L2:以菌株P3的基因组为模板，L3:以突变株P3ΔpykA的基因组为模板。
通过在KB培养基进行液体培养，同时测定菌株P3和P3ΔpykA生长曲线，如图22所示。结果表明，在菌株P3中敲除pykA基因不会影响其生长，表明此基因工程菌株稳定性非常好，有利于其工业化应用。
实施例9
本实施例涉及一种利用实施例7制备的基因工程菌株合成PCN的方法，包括如下步骤：
分别将菌株HT66ΔpykA和HT66wt(野生株HT66作为对照)分别在KB固体平板上活化两次，然后接至5mL KB液体培养基培养，培养至对数期，以2％的比例转接至装有60mL KB培养基的三角瓶进行发酵。发酵条件为28℃，180rpm。定时取样，以HPLC测定菌株PCN产量。
HPLC检测条件：
流动相为乙腈-25mM乙酸铵，色谱柱为WondaSilC18-WRreversephasecolumn(5μm；4.6×250mm,Shimadzu,Japan)，检测波长254nm，检测条件：0-2min，8％乙腈-92％25mM乙酸铵，2-20min，乙腈浓度从8％升至60％，乙酸铵浓度从92％下降至40％，20-21min，8％乙腈-92％25mM乙酸铵。
分别测定菌株HT66和菌株HT66ΔpykA的PCN产量，结果如图23所示。从图可 见，敲除pykA基因可以明显提高PCN产量，对照株HT66的PCN产量为552mg/L，而突变株在48h时其PCN产量为1038mg/L，相比提高了88％。由此可见，在绿针假单胞菌HT66中敲除pykA基因能够有效改变物质的流向，有利于积累PCN。
实施例10
本实施例涉及一种利用实施例8制备的基因工程菌株生产PCN的方法，包括如下步骤：
首先将菌株P3ΔpykA和P3(对照)分别在KB固体平板上活化，然后将其接至5mL KB液体培养基培养，待其生长到对数期后分别接种至装有60mL KB培养基的三角瓶进行发酵。初始接种量OD600为0.02。发酵条件为28℃，180rpm。定时取样，分别测定菌株P3ΔpykA和P3发酵液中的PCN产量，结果如图24所示。
从图可见，敲除pykA基因可以明显提高PCN产量，在36h时其PCN产量为3594mg/L，与同样条件下对照株P3的PCN产量(1923mg/L)相比，提高了71.3％。该结果表明，在绿针假单胞菌P3中敲除pykA基因能够有效的再分配细胞碳、氮源的流向，使其流向莽草酸途径，进而流向PCN合成。
本发明具体是以绿针假单胞菌HT66为出发菌株，通过基因工程技术从HT66菌株及其已知突变株基因组中分别或者同时敲除吩嗪合成过程中的lon、parS负调控基因，构建lon、parS基因单基因突变株或双基因工程菌株，使得吩嗪-1-甲酰胺(phenazine-1-carboxamide,CAS号为550-89-0，简称PCN)的产量大幅提高，能够更加有效由于生物防治。
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变形或修改，这并不影响本发明的实质内容。

Claims (13)

  1. 一株高产吩嗪-1-甲酰胺的基因工程菌株，其特征在于，具体是敲除绿针假单胞菌(Pseudomonas chlororaphis)HT66 CCTCC NO:M 2013467及其衍生物基因组的lon基因、parS基因中的一个或两个基因而得到基因工程菌株；
    或者，敲除绿针假单胞菌(Pseudomonas chlororaphis)HT66 CCTCC NO:M 2013467rpeA、psrA单突变株或者双突变株基因组的lon基因、parS基因中的一个或两个基因而得到基因工程菌株；其中，所述单突变株为HT66ΔrpeA、HT66ΔpsrA，双突变株为HT66ΔrpeAΔpsrA；
    或者敲除了绿针假单胞菌(Psedunomonaschlororaphis)HT66 CCTCC NO:M2013467及其衍生物基因组中的pykA基因，即得。
  2. 根据权利要求1所述的高产吩嗪-1-甲酰胺的基因工程菌株，其特征在于，所述lon基因的碱基序列如SEQ ID NO.1所示；所述parS基因的碱基序列如SEQ ID NO.2所示，所述pykA基因的碱基序列如SEQ ID NO.11所示，所述pykA基因的对应蛋白质的氨基酸序列如SEQ ID NO.12所示。
  3. 根据权利要求1所述的高产吩嗪-1-甲酰胺的基因工程菌株，其特征在于，所述基因工程菌株包括HT66Δlon、HT66ΔparS、HT66ΔlonΔparS、HT66ΔlonΔrpeA、HT66ΔparSΔrpeA、HT66ΔlonΔpsrA、HT66ΔpsrAΔparS、HT66ΔlonΔparSΔrpeA、HT66ΔlonΔparSΔpsrA、HT66ΔlonΔpsrAΔrpeA、HT66ΔparSΔpsrAΔrpeA、HT66ΔlonΔparSΔpsrAΔrpeA、HT66ΔpykA。
  4. 一种根据权利要求1所述高产吩嗪-1-甲酰胺的基因工程菌株的构建方法，其特征在于，具体是：敲除绿针假单胞菌(Pseudomonas chlororaphis)HT66 CCTCC NO:M 2013467及其衍生物基因组的lon基因、parS基因中的一个基因或两个基因，即可；
    或者，敲除绿针假单胞菌(Pseudomonas chlororaphis)HT66CCTCC NO:M 2013467rpeA、psrA单突变株或者双突变株基因组的lon基因、parS基因中的一个或两个基因而得到基因工程菌株；其中，所述单突变株为HT66ΔrpeA、HT66ΔpsrA，双突变株为HT66ΔrpeAΔpsrA；
    或者，敲除了绿针假单胞菌(Psedunomonaschlororaphis)HT66 CCTCC NO:M2013467及其衍生物基因组中的pykA基因，即得。
  5. 根据权利要求4所述高产吩嗪-1-甲酰胺的基因工程菌株的构建方法，其特征在 于，所述构建方法包括：
    i、扩增lon基因或parS基因片段的上下游同源臂；
    ii、采用融合PCR法连接上下游同源臂并插入pK18mobsacB质粒中得lon基因或parS基因重组质粒；
    iii、(a)使lon基因重组质粒上的lon基因上下游同源臂片段与HT66、HT66ΔparS、HT66ΔrpeA、HT66ΔrpeAΔparS、HT66ΔpsrA、HT66ΔpsrAΔparS、HT66ΔrpeAΔpsrA、HT66ΔrpeAΔpsrAΔparS基因组中的lon基因发生同源重组；
    或(b)使parS基因重组质粒上的parS基因上下游同源臂片段与HT66、HT66Δlon、HT66ΔrpeA、HT66ΔrpeAΔlon、HT66ΔpsrA、HT66ΔpsrAΔlon、HT66ΔrpeAΔpsrA、HT66ΔrpeAΔpsrAΔlon基因组中的parS基因发生同源重组；
    筛选阳性克隆，即得；
    或，敲除所述pykA基因的方法为插入突变法或无痕敲除法；
    所述插入突变法敲除pykA基因包括如下步骤：
    S1、扩增pykA基因片段，并插入pEX18Tc质粒中；
    S2、扩增卡那霉素或庆大霉素等抗性基因，插入pykA基因中间，使pykA基因发生插入突变；
    S3、将突变后的pykA基因与HT66基因组中的pykA基因发生同源重组，根据抗性筛选出阳性克隆，即可；
    所述无痕敲除pykA基因包括如下步骤：
    A1、扩增pykA基因的上下游同源臂；
    A2、融合PCR法连接上下游同源臂并插入pK18mobsacB质粒中；
    A3、使重组质粒上的pykA基因的上下游同源臂片段与HT66基因组发生同源重组，利用蔗糖压力和抗性筛选出阳性克隆，即可。
  6. 一种基于权利要求1至3任一项所述基因工程菌株的吩嗪-1-甲酰胺合成方法，其特征在于，具体包括如下步骤：将所述基因工程菌株的种子发酵液接种于KB发酵培养基中培养，即可。
  7. 根据权利要求6所述的基因工程菌株的吩嗪-1-甲酰胺合成方法，其特征在于，所述种子发酵液的制备具体为：将所述基因工程菌株接种于KB培养基中，置于28℃、180转/分下培养至OD600为0.5～2.0，即得。
  8. 根据权利要求6所述的基因工程菌株的吩嗪-1-甲酰胺合成方法，其特征在于， 所述培养的条件具体为：26～34℃、100～300转/分钟、24～72h。
  9. 根据权利要求6所述的基因工程菌株的吩嗪-1-甲酰胺合成方法，其特征在于，所述种子发酵液与所述KB发酵培养基的体积比为(1～10):100。
  10. 一种基于权利要求1至3任一项所述基因工程菌株的吩嗪-1-甲酰胺合成方法，其特征在于，具体包括如下步骤：将所述基因工程菌株接种于KB液体培养基中培养，即可。
  11. 一种用于培养权利要求10所述的基因工程菌株的培养基，其特征在于，包括按重量份数计的如下组分：
    Figure PCTCN2017098480-appb-100001
  12. 一种如权利要求1～3中任意一项所述的高产吩嗪-1-甲酰胺的基因工程菌株在生产吩嗪-1-甲酰胺中的用途。
  13. 如权利要求12所述的用途，其中，所述基因工程菌株在制备吩嗪-1-甲酰胺时的条件为：好氧培养；温度：26～34；pH：6～8；转速100～350rpm。
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