CN112094795B - Pseudomonas chlororaphis QHPHZ-8 for producing 1-hydroxyphenyloxazine and application - Google Patents

Pseudomonas chlororaphis QHPHZ-8 for producing 1-hydroxyphenyloxazine and application Download PDF

Info

Publication number
CN112094795B
CN112094795B CN202011024003.0A CN202011024003A CN112094795B CN 112094795 B CN112094795 B CN 112094795B CN 202011024003 A CN202011024003 A CN 202011024003A CN 112094795 B CN112094795 B CN 112094795B
Authority
CN
China
Prior art keywords
pseudomonas chlororaphis
pseudomonas
strain
gene
chlororaphis
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
Application number
CN202011024003.0A
Other languages
Chinese (zh)
Other versions
CN112094795A (en
Inventor
刘开泉
李玲
王瑞明
王腾飞
李丕武
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Qilu University of Technology
Original Assignee
Qilu University of Technology
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Qilu University of Technology filed Critical Qilu University of Technology
Priority to CN202011024003.0A priority Critical patent/CN112094795B/en
Publication of CN112094795A publication Critical patent/CN112094795A/en
Priority to AU2021103349A priority patent/AU2021103349A4/en
Priority to ZA2021/07098A priority patent/ZA202107098B/en
Application granted granted Critical
Publication of CN112094795B publication Critical patent/CN112094795B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
    • A01N43/60—1,4-Diazines; Hydrogenated 1,4-diazines
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/27—Pseudomonas
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/36—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems
    • C07D241/38—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings condensed with carbocyclic rings or ring systems with only hydrogen or carbon atoms directly attached to the ring nitrogen atoms
    • C07D241/40—Benzopyrazines
    • C07D241/44—Benzopyrazines with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the hetero ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Pest Control & Pesticides (AREA)
  • Dentistry (AREA)
  • Agronomy & Crop Science (AREA)
  • Environmental Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention particularly relates to pseudomonas chlororaphis QHPHZ-8 for producing 1-hydroxyphenyloxazine and application thereof. The invention obtains a strain of Pseudomonas chlororaphis Qlu-1 with typical gene cluster synthesized by phenazine compounds through screening, and the strain can not synthesize and secrete 1-OH-PHZ naturally. The invention firstly prepares QOHPHZ-1 by replacing phzO gene with phzS, and obtains 1-OH-PHZ accumulation. In order to further obtain a 1-hydroxyphenyloxazine high-yield strain, the invention carries out subsequent optimization work aiming at the QOHPHZ-1 strain to prepare the QOHPHZ-8 strain, the yield of 1-OH-PHZ in unit time is improved by 28 times, and the strain is expected to become an engineering strain applied to the fields of agricultural production and algae prevention and control.

Description

Pseudomonas chlororaphis QHPHZ-8 for producing 1-hydroxyphenyloxazine and application
Technical Field
The invention belongs to the technical field of microbial engineering strains, and particularly relates to Pseudomonas chlororaphis (QHPHZ-8) for producing 1-hydroxy-phenazine (1-OH-PHZ), and application of the strain as an engineering strain.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The pesticide is an important material for agricultural production and has important significance for maintaining the agricultural ecological environment. Compared with chemical pesticides, the biological pesticide has the advantages of safety to non-target organisms, small toxic and side effects, good compatibility to the environment and the like, and has a great number of unique advantages and good development prospects. Meanwhile, biopesticides face development bottlenecks, such as low fermentation unit of biocontrol pseudomonas.
Phenazine is a nitrogen-containing heterocyclic pigment, and it has been found that almost all phenazine compounds exhibit biocontrol activity against bacteria and fungi, and that the major antibiotic resistance in Pseudomonas bacteria includes phenazine-1 carboxylic acid (PCA), 1-OH-PHZ, 2-hydroxyphenylazine (2-OH-PHZ), Pyocyanamide (PYO), phenazine-1-carboxamide (PCN), and the like.
1-OH-PHZ is a secondary metabolite of Pseudomonas and causes cell death by increasing intracellular oxygen free radical production. The substance has good stability, and is expected to exert stable sterilization and disease prevention effects when being applied as a microbial pesticide. In addition, researches show that the 1-OH-PHZ also has good inhibition effect on aspergillus terreus, green algae and blue algae, and is expected to be applied to marine pollution prevention and treatment. At present, the synthesis method of 1-OH-PHZ mainly comprises a biosynthesis pathway and a chemical synthesis pathway. According to literature reports, the chemical synthesis route of 1-OH-PHZ includes the following two: (1) the 1-OH-PHZ is prepared by the cycloaddition reaction of phenyl-benzofuran oxide and cyclohexanedione to obtain the nitrogen oxide of 1-OH-PHZ and then reducing the nitrogen oxide part by sodium dithionite. (2) Haddadin et al also propose reacting a phenyl-and-furan oxide with 1, 2-cyclohexanedione under triethylamine, nitrogen conditions for 7 to 8 hours to obtain a dark brown reaction mixture; the mixture is acidified to obtain a mixture of 1-hydroxyphenyloxazine nitroxides, which is then treated with sodium dithionite to obtain 1-OH-PHZ. In the aspect of biosynthesis pathway, the current research shows that the synthesis of 1-OH-PHZ is mainly related to the shikimic acid pathway in pseudomonas, shikimic acid is converted into chorismic acid, phenazine-1-carboxylic acid is further synthesized, and finally the shikimic acid is converted into 1-OH-PHZ under the action of PhzS protein. Based on the advantages of the biosynthetic synthetic route, engineering strains with higher yield are obtained by modifying pseudomonas strains, so that the method has higher research value.
Based on the previous research results of the inventor, the Pseudomonas chlororaphis (Pseudomonas chlororaphis) contains a core gene component for synthesizing phenazine substances and a phenazine related modifier gene, and the Pseudomonas chlororaphis (Pseudomonas chlororaphis) has low pathogenicity and is safe to use. Because the chemical synthesis conditions of the 1-OH-PHZ are harsh and the environment is not friendly, the method for obtaining the engineering strain with high yield of the 1-OH-PHZ based on the microorganism has important significance.
Disclosure of Invention
Based on the above description in the background art, the present invention first obtained a Pseudomonas chlororaphis (Pseudomonas chlororaphis) Qlu-1 strain by screening, which has the typical gene cluster phzABCBEFG for phenazine synthesis and can produce phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenylazine (2-OH-PHZ). However, natural Pseudomonas chlororaphis Qlu-1 cannot synthesize 1-OH-PHZ. According to the invention, the phenazine modifying gene phzO in Qlu-1 is replaced by the phenazine modifying gene phzS to obtain the Pseudomonas chlororaphis (Pseudomonas chlororaphis) QHPHZ-1, so that the strain can express 1-OH-PHZ, and the highest yield of the 1-OH-PHZ of the Pseudomonas chlororaphis (Pseudomonas chlororaphis) QHPHZ-1 is about 20mg/L, so that the application requirement can not be met. The invention further carries out gene transformation on QOHPHZ-1 to obtain the Pseudomonas chlororaphis (QOHPHZ-8), the yield can reach about 560mg/L, and is improved by 28 times, and the invention is expected to be used as an engineering strain for agricultural production and algae control.
Based on the technical effects, the invention provides the following technical scheme:
the invention provides a Pseudomonas chlororaphis (Pseudomonas chlororaphis) QHPHZ-1 for producing 1-hydroxy-phenazine, wherein the Pseudomonas chlororaphis QHPHZ-1 is obtained by using Pseudomonas chlororaphis (Pseudomonas chlororaphis) Qlu-1 as an initial strain and replacing a gene phzO in Qlu-1 with an exogenous phzS gene; the starting strain Pseudomonas chlororaphis Qlu-1 is preserved in China Center for Type Culture Collection (CCTCC) at 05-8.2020 with the address as follows: china, Wuhan and Wuhan university, the biological preservation number is: CCTCC NO: m2020108.
Based on the method described in the first aspect, Pseudomonas chlororaphis Qlu-1 was modified to express 1-OH-PHZ, and the first of all, it was realized that there is no way to have it. The invention further carries out subsequent optimization work aiming at the strain to obtain the high-yield strain of Pseudomonas chlororaphis (QHPHZ-8) of 1-OH-PHZ.
In a second aspect of the present invention, there is provided a Pseudomonas chlororaphis (Pseudomonas chlororaphis) QHPHZ-8 for producing 1-OH-PHZ, wherein said Pseudomonas chlororaphis QHPHZ-8 has a genome insert copy of the gene fragment of tktA-ppsA-phzC-aroB-aroD-aroE and a deletion of the genes pykF, lon, psrA, parS, rsmE, rpeA, etc., as a starting strain.
In a third aspect of the present invention, there is provided the use of Pseudomonas chlororaphis QHPHZ-1 of the first aspect and/or Pseudomonas chlororaphis QHPHZ-8 of the second aspect as a 1-hydroxyphenyloxazine-producing engineering strain.
In a fourth aspect of the present invention, there is provided a microbial preparation comprising one or a mixture of the strains of Pseudomonas chlororaphis QHPHZ-1 of the first aspect, and Pseudomonas chlororaphis QHPHZ-8 of the second aspect, or a culture thereof.
In a fifth aspect of the invention, the application of the microbial inoculum of the fourth aspect in preparing agricultural production products is provided.
In a sixth aspect of the present invention, there is provided an algae control preparation comprising the microbial agent of the fourth aspect.
The beneficial effects of one or more technical schemes are as follows:
1. the pathogenicity of the pseudomonas chlororaphis is lower, the pseudomonas chlororaphis used for engineering strains is safer compared with the existing pseudomonas aeruginosa, and the optimization method provided by the invention obviously improves the yield of the modified strains aiming at the defect that the fermentation amount of the existing biosynthesis route cannot meet the application requirement.
2. Research in the art on the synthetic pathway of phenazines in P.chlororaphis has shown that the same target is often associated with multiple physiological activities, and that simultaneous alteration of multiple targets may lead to unexpected results. The gene editing mode provided by the invention effectively improves the yield of 1-OH-PHZ and has reference significance for further determining the synthetic mechanism of phenazine substances in microorganisms.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is an upstream and downstream amplification of the psrA gene at position 1 in example 2;
in the figure 1(A), 1 is DNA Ladder,2-3 is psrA upstream amplification sequence, and 4-5 is psrA downstream amplification sequence;
in FIG. 1(B), 1 is DNA Ladder, and 2-3 are psrA upstream and downstream fusion fragments.
FIG. 2 shows the results of the screening of the psrA gene parental hybrid double-resistant plate in example 2.
FIG. 3 is a photograph of example 2 screening for double crossover positive clones of the psrA gene.
FIG. 4 is a PCR validation of the psrA knockout in example 2;
detection of an external primer: 1, taking a psrA knockout strain genome as a template amplification fragment; 2, DNA Ladder; 3, blank control; 4, using the genome of the wild strain as a template amplification fragment;
and (3) detecting an inner primer: 1, using the genome of a wild strain as a template amplification fragment; 2, DNA Ladder; 3, taking the genome of the psrA knockout strain as a template amplification fragment; 4, blank control.
FIG. 5 is a graph of 1-OH-PHZ yield for different strains.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As introduced in the background art, the prior art has the defect of low yield of 1-OH-PHZ biosynthesis pathway, and in order to solve the technical problems, the invention provides Pseudomonas chlororaphis (QHPHZ-8) with high yield of 1-OH-PHZ.
The invention provides a Pseudomonas chlororaphis (Pseudomonas chlororaphis) QHPHZ-1 for producing 1-hydroxy-phenazine, wherein the Pseudomonas chlororaphis QHPHZ-1 is obtained by using Pseudomonas chlororaphis (Pseudomonas chlororaphis) Qlu-1 as an initial strain and replacing a gene phzO in Qlu-1 with an exogenous phzS gene; the starting strain Pseudomonas chlororaphis Qlu-1 is preserved in China Center for Type Culture Collection (CCTCC) at 05-8.2020 with the address as follows: china, Wuhan and Wuhan university, the biological preservation number is: CCTCC NO: m2020108.
Editing of related gene fragments in Pseudomonas chlororaphis (Pseudomonas chlororaphis) strains provided by the first aspect and the second aspect of the invention can clip related genes by gene editing means commonly used in the art, and the gene phzO knockout or exogenous phzS gene introduction mode comprises but is not limited to gene editing by a siRNA gene silencing mechanism, Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENS), CRISPER-Cas9 system, a lentiviral vector or plasmid transfection mode.
Further preferably, in one embodiment of the present invention, the gene knockout or introduction is achieved by means of plasmid transfection.
In a second aspect of the present invention, there is provided a Pseudomonas chlororaphis (Pseudomonas chlororaphis) QHPHZ-8 for producing 1-OH-PHZ, wherein said Pseudomonas chlororaphis QHPHZ-8 has a genome insert copy of the gene fragment of tktA-ppsA-phzC-aroB-aroD-aroE and a deletion of the genes pykF, lon, psrA, parS, rsmE, rpeA, etc., as a starting strain.
In a third aspect of the present invention, there is provided the use of Pseudomonas chlororaphis QHPHZ-1 of the first aspect and/or Pseudomonas chlororaphis QHPHZ-8 of the second aspect as a 1-OH-PHZ producing engineered strain.
In a fourth aspect of the present invention, there is provided a microbial preparation comprising one or a mixture of the strains of Pseudomonas chlororaphis QHPHZ-1 of the first aspect, and Pseudomonas chlororaphis QHPHZ-8 of the second aspect, or a culture thereof.
Preferably, the culture includes, but is not limited to, a fermentation of the strain, or a metabolite isolated after fermentation of the strain.
Preferably, the formulation of the microbial inoculum includes but is not limited to liquid, powder, granules or tablets.
Preferably, the microbial inoculum is wettable powder, water dispersible granules or water suspending agent.
Preferably, the microbial inoculum also comprises necessary auxiliary materials.
In a fifth aspect of the invention, the application of the microbial inoculum of the fourth aspect in preparing agricultural production products is provided.
Preferably, the agricultural production articles include, but are not limited to, pesticides, fertilizers, culture media, and the like.
Further preferably, the pesticide uses the microbial inoculum of the fifth aspect as an active ingredient, or is applied in combination with other pesticides, microbial pesticides and the like.
Further preferably, the fertilizer includes, but is not limited to, an ammonium phosphate fertilizer, a macroelement water-soluble fertilizer, a secondary element fertilizer, a biological fertilizer, an organic fertilizer or a multi-dimensional energy-concentrated organic fertilizer.
Further preferably, the culture medium is a greenhouse seedling culture medium.
In a sixth aspect of the present invention, there is provided an algae control preparation comprising the microbial agent of the fourth aspect.
The application purpose of the algae control preparation comprises but is not limited to the aspects of ecological wetland treatment, sewage treatment, algae removal in the breeding industry, marine pollution treatment and the like, and the existing research proves that the 1-OH-PHZ also has good inhibition effect on partial algae, and has better environmental compatibility when being used as an environmental control reagent compared with the traditional chemical reagent.
In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the technical scheme of the present invention will be described in detail with reference to specific examples, and reagents designed in the following examples are all commercially available products unless otherwise specified.
Example 1
In the present embodiment, there is provided a method for preparing Pseudomonas chlororaphis (Pseudomonas chlororaphis) QOHPHZ-1 based on Pseudomonas chlororaphis (Qlu-1), the steps of the preparation method are as follows:
1. inoculating Pseudomonas chlororaphis Qlu-1 into KB (A +) culture medium, shaking and culturing overnight at 30 ℃ with shaking table at 180rpm, extracting Qlu-1 genome by using genome extraction kit, and preserving at-20 ℃ for later use.
2. Searching phzO gene and upstream and downstream sequences of the phzO gene in sequenced Qlu-1 genome data, and amplifying an upstream fragment phzO-U and a downstream fragment phzO-D of the phzO gene by using a Qlu-1 strain genome as a template and using phzO-F1/phzO-R1 and phzO-F2/phzO-R2 as primers respectively; and (3) amplifying the upstream and downstream fusion fragments phzO-UD of phzO by using phzO-U and phzO-D as templates and phzO-F1/phzO-R2 as templates. And constructing a recombinant plasmid pk18-phzO-UD by using the fusion fragment phzO-UD and the knock-out plasmid pk18mobsacB through an enzyme digestion connection method.
By heat shock transformation, pk18-phzO-UD was introduced into E.coli S17-1 (. lamda.), E.coli S17-1 (. lamda.) was subjected to parental cross culture with Pseudomonas Qlu-1, and recombinant plasmid pk18-phzO-UD was introduced into Pseudomonas Qlu-1. Pseudomonas aeruginosa Qlu-1 was co-cultured with E.coli S17-1 (. lamda.) and plated on KB (A + K +) double antibody plates to select single colonies. Jointly screening Qlu-1 phzO knockout strains by methods such as sucrose plate screening, photocopy screening, PCR screening and the like, detecting by HPLC after fermentation, accumulating phenazine-1-carboxylic acid in fermentation liquor, and naming the strains as QPCA.
Using Qlu-1 genome as a template, using phzO-F1/phzO-R1-2 and phzO-F2-2/phzO-R2 as primers to amplify an upstream fragment phzO-U2 and a downstream fragment phzO-D2 of the phzO gene, using the synthesized phzS gene as a template, using phzS-F1/phzS-R1 as primers to amplify a fragment phzS-2, using phzO-U2, phzO-D2 and phzS-2 as templates, using phzO-F1/phzO-R2 as a template to amplify a phzS introduction fragment phzS-IN fused with the upstream and downstream of the phzO gene. Constructing a recombinant plasmid pk18-phzS-IN by the fusion fragment phzS-IN and the knock-out plasmid pk18mobsacB through an enzyme digestion connection method, and introducing the recombinant plasmid pk18-phzS-IN into escherichia coli S17-1 (lambda) IN a heat shock transformation mode. Coli S17-1 (. lamda.) was subjected to parental hybridization with Pseudomonas Qlu-1, and the recombinant plasmid pk18-phzS-IN was introduced into Pseudomonas QPCA.
Qlu-1phzS insertion strains are jointly screened by methods such as sucrose plate screening, photocopy screening, PCR screening and the like, HPLC detection is carried out after fermentation, only 1-OH-PHZ is accumulated in fermentation liquor after 48 hours, and the strain is named as QOHPHZ-1. After QOHPHZ-1 is fermented, the QOHPHZ-1 can generate 20.4 mg/L1-OH-PHZ at 96h by HPLC detection after ethyl acetate extraction.
Example 2
In this example, there is provided a process for producing Pseudomonas chlororaphis (Pseudomonas chlororaphis) based on Pseudomonas chlororaphis (QHPHZ-1) described in example 1
A method of QOHPHZ-8, said preparation method comprising the steps of:
under the condition of QOHPHZ-1, phenazine synthesis leader pathway related genes phzC, aroB, aroD and aroE and central metabolism related genes tktA and ppsA are respectively amplified. And a Bglric modular plasmid pBbB8k-GFP is used as a medium plasmid to respectively construct a single modular plasmid, and the genes are integrated into a tandem gene pBbB8K-tktA-ppsA-phzC-aroB-aroD-aroE by using a modular principle.
The tandem gene is cut from pBbB8K-tktA-ppsA-phzC-aroB-aroD-aroE, is connected to an integration plasmid pUC18-mini-Tn7T-Gm, and is integrated into a QHPHZ-1 genome through a pUC18-mini-Tn7T-Gm plasmid under the assistance of an auxiliary plasmid pTNS2 to obtain a strain QHPHZ-2 (Pseudomonas chlororaphis) which is found to achieve the yield of 83.8mg/L of 1-OH-PHZ in 96h after fermentation.
The gene pykF, lon, psrA, parS and rsmE rpeA in Pseudomonas chlororaphis (Pseudomonas chlororaphis) QHPHZ-2 is used as an initial strain to knock out the gene pykF, lon, psrA, parS and rsmE rpeA.
Genetic manipulation is described by way of example for a traceless knockout of the psrA gene. Qlu-1 genome is extracted by using the kit, the psrA gene and the sequence upstream and downstream sequences thereof are searched in the sequenced Qlu-1 genome sequence, and the upstream and downstream fragments of the psrA gene are fished and connected by PCR: psrA-ud.
The psrA-ud fragment was ligated to plasmid pK18mobsacB by a seamless cloning technique to construct the psrA knock-out plasmid pK18-psrA-ud, which was introduced into E.coli S17-1 (. lamda.) by heat shock transformation.
Pseudomonas chlororaphis QHPHZ-4 and Escherichia coli S17-1 (lambda) are co-cultured and then coated on a KB (A + K +) double-antibody plate, a single colony is selected and coated on a 15% sucrose KB plate containing ampicillin, and a QHPHZ-4 knockout psrA strain is screened and verified through PCR.
By the same method, negative regulatory genes pykF, lon, psrA, parS, rsmE and rpeA are sequentially knocked out from a QOHZ-2 genome to obtain a high-yield 1-OH-PHZ strain QOHZ-8, and after fermentation, extraction by ethyl acetate and HPLC detection, the QOHZ-8 can generate 560 mg/L1-OH-PHZ at 96 h.
FIGS. 1-4 illustrate the above steps by way of example for a psrA knockout. In FIG. 5, the genes of pykF, lon, psrA, par, rsmE and rpeA of Pseudomonas aeruginosa QOHPHZ-2 which have been knocked out in sequence are designated QOHPHZ-3, QOHPHZ-4, QOHPHZ-5, QOHPHZ-6, QOHPHZ-7 and QOHPHZ-8, respectively. The yields of 1-OH-PHZ of the QOHPHZ-3, QOHPHZ-4, QOHPHZ-5, QOHPHZ-6, QOHPHZ-7 and QOHPHZ-8 strains are shown in FIG. 5.
Comparative example 1
In this example, a mode of expressing related genes by plasmid expression is provided, in which the genes are first constructed into pBbB8K-GFP plasmids, the single-gene plasmids are integrated into the tandem genes pBbB8K-tktA-ppsA-phzC-aroB-aroD-aroE by Bglviral modular plasmid operating rules, the recombinant plasmids are then introduced into the strain QOHZ-1 by electric shock transformation to obtain the strain QOHZ-2-2, fermentation in KB supplemented with Kan antibiotics reveals that QOHZ-2-2 also has 1-OH-PHZ, and the yield is 52.6mg/L, and compared with plasmid expression, genome integration expression is not only stable, but also has higher efficiency
(1-OHPHZ yield of tktA-ppsA-phzC-aroB-aroD-aroE genome integration strain QHPHZ-2 83.8 mg/L).
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (12)

1. Pseudomonas chlororaphis (A) for producing 1-hydroxy-phenazinePseudomonas chlororaphis) QOHZ-1, wherein said Pseudomonas chlororaphis QOHPHZ-1 is Pseudomonas chlororaphis (QOHPHZ-1)Pseudomonas chlororaphis) Qlu-1 as original strain, adopting exogenous sourcephzSGene substitution Qlu-1phzOObtaining; the starting strain Pseudomonas chlororaphis (A)Pseudomonas chlororaphis) Qlu-1 has been deposited in China Center for Type Culture Collection (CCTCC) at 8.05.2020 with addresses of: china, Wuhan and Wuhan university, the biological preservation number is: CCTCC NO: m2020108.
2. Pseudomonas chlororaphis (Pseudomonas aeruginosa) for producing 1-hydroxy-phenazine according to claim 1Pseudomonas chlororaphis) QOHZ-1, wherein the gene isphzOKnock-out or exogenousphzSThe gene introduction mode is to carry out gene editing through an siRNA gene silencing mechanism, zinc finger nuclease, transcription activator like effector nuclease, CRISPER-Cas9 system, a lentiviral vector or plasmid transfection mode.
3. Pseudomonas chlororaphis (Pseudomonas aeruginosa) for producing 1-hydroxy-phenazine according to claim 2Pseudomonas chlororaphis) QOHZ-1, wherein said gene knockout or introduction is effected by means of plasmid transfection.
4. Pseudomonas chlororaphis (A) for producing 1-OH-PHZPseudomonas chlororaphis) QOHZ-8, characterized in that said Pseudomonas chlororaphis QOHZ-8 is the Pseudomonas chlororaphis of any one of claims 1 to 3 (A), (B), (C), (D) and (D)Pseudomonas chlororaphis) QOHZ-1 as starting strain havingtktA-ppsA-phzC-aroB-aroD-aroEMultiple copies of gene fragment genome insert, simultaneous genepykF, lon, psrA, parS, rsmE,rpeAIs absent.
5. Pseudomonas chlororaphis (Pseudomonas aeruginosa) according to any one of claims 1 to 3Pseudomonas chlororaphis) QOHZ-1 and/or Pseudomonas chlororaphis (Pseudomonas aeruginosa) of claim 4Pseudomonas chlororaphis) Application of QOHZ-8 as a 1-hydroxyphenyloxazine production engineering strain.
6. A microbial preparation comprising Pseudomonas chlororaphis (M) as defined in any one of claims 1 to 3Pseudomonas chlororaphis) QOHZ-1, Pseudomonas chlororaphis (Pseudomonas aeruginosa) of claim 4Pseudomonas chlororaphis) One or a mixture of QOHZ-8.
7. The microbial inoculum of claim 6, which is in the form of liquid, powder, granules or tablets.
8. The microbial inoculum of claim 7, wherein the microbial inoculum is in the form of wettable powder, water dispersible granules or water suspending agent.
9. Use of the fungicide according to any one of claims 6 to 8 for the preparation of an agricultural article of manufacture, characterized in that said agricultural article of manufacture is a pesticide, a fertilizer or a culture medium.
10. Use of the fungicide according to claim 9 for the preparation of agricultural products, characterized in that the pesticide comprises the fungicide according to any one of claims 6 to 8 as an active ingredient.
11. Use of the inoculant according to claim 9 for the preparation of an agricultural produce, wherein the fertilizer is an ammonium phosphate fertilizer, a macroelement water soluble fertilizer, a secondary element fertilizer, a biological fertilizer, an organic fertilizer or a multi-dimensional energy-concentrated organic fertilizer.
12. The use of the microbial inoculum of claim 9 in the preparation of agricultural products, wherein the culture medium is a greenhouse seedling culture medium.
CN202011024003.0A 2020-09-25 2020-09-25 Pseudomonas chlororaphis QHPHZ-8 for producing 1-hydroxyphenyloxazine and application Active CN112094795B (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN202011024003.0A CN112094795B (en) 2020-09-25 2020-09-25 Pseudomonas chlororaphis QHPHZ-8 for producing 1-hydroxyphenyloxazine and application
AU2021103349A AU2021103349A4 (en) 2020-09-25 2021-06-15 Pseudomonas Chlororaphis QOHPHZ-8 for producing 1-Hydroxy-Phenazine (1-OH-PHZ) and use thereof
ZA2021/07098A ZA202107098B (en) 2020-09-25 2021-09-23 Pseudomonas chlororaphis qohphz-8 for producing 1-hydroxy-phenazine (1-oh-phz) and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202011024003.0A CN112094795B (en) 2020-09-25 2020-09-25 Pseudomonas chlororaphis QHPHZ-8 for producing 1-hydroxyphenyloxazine and application

Publications (2)

Publication Number Publication Date
CN112094795A CN112094795A (en) 2020-12-18
CN112094795B true CN112094795B (en) 2022-02-22

Family

ID=73756232

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202011024003.0A Active CN112094795B (en) 2020-09-25 2020-09-25 Pseudomonas chlororaphis QHPHZ-8 for producing 1-hydroxyphenyloxazine and application

Country Status (3)

Country Link
CN (1) CN112094795B (en)
AU (1) AU2021103349A4 (en)
ZA (1) ZA202107098B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114574505A (en) * 2022-03-17 2022-06-03 上海交通大学 Monooxygenase gene phzO, encoded protein, genetic engineering strain, construction method and application thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102399737B (en) * 2011-11-07 2015-04-08 上海交通大学 Biological engineering bacterial strain for producing novel microorganism source fungicide and application of biological engineering bacterial strain
CN103642714B (en) * 2013-11-13 2015-10-28 上海交通大学 For the production of the engineering strain and uses thereof of azophenlyene-1-methane amide
CN103602621B (en) * 2013-12-02 2015-04-15 江苏省农业科学院 Pseudomonas choloeaphtis and application

Also Published As

Publication number Publication date
AU2021103349A4 (en) 2021-08-05
ZA202107098B (en) 2022-03-30
CN112094795A (en) 2020-12-18

Similar Documents

Publication Publication Date Title
Sun et al. Bacillus velezensis stimulates resident rhizosphere Pseudomonas stutzeri for plant health through metabolic interactions
Worsley et al. Streptomyces endophytes promote host health and enhance growth across plant species
Schlechter et al. Chromatic bacteria–A broad host-range plasmid and chromosomal insertion toolbox for fluorescent protein expression in bacteria
Pothier et al. Promoter-trap identification of wheat seed extract-induced genes in the plant-growth-promoting rhizobacterium Azospirillum brasilense Sp245
Fenton et al. Manipulating each MreB of Bdellovibrio bacteriovorus gives diverse morphological and predatory phenotypes
CN106635937B (en) Genetic engineering strain for high-yield phenazine-1-formamide and construction method thereof
CN102399737A (en) Biological engineering bacterial strain for producing novel microorganism source fungicide and application of biological engineering bacterial strain
CN101182469B (en) Aspergillus niger strain having highly-resistant activity to carbendazol and uses thereof
CN110257420A (en) Plant gene silencing carrier and its construction method and application based on CasRx
Shanmugam et al. The CRISPR/Cas9 system for targeted genome engineering in free-living fungi: advances and opportunities for lichenized fungi
El Othmany et al. Current understanding on adhesion and biofilm development in actinobacteria
Vandepol et al. Linnemannia elongata (Mortierellaceae) stimulates Arabidopsis thaliana aerial growth and responses to auxin, ethylene, and reactive oxygen species
CN112094795B (en) Pseudomonas chlororaphis QHPHZ-8 for producing 1-hydroxyphenyloxazine and application
Zhang et al. Colonization of Beauveria bassiana 08F04 in root-zone soil and its biocontrol of cereal cyst nematode (Heterodera filipjevi)
Morales-Cedeño et al. Functional and genomic analysis of Rouxiella badensis SER3 as a novel biocontrol agent of fungal pathogens
Bouneb et al. Occurrence and transmission of mycovirus Cryphonectria hypovirus 1 from dejecta of Thyreophagus corticalis (Acari, Acaridae)
Almeida et al. Unveiling concealed functions of endosymbiotic bacteria harbored in the ascomycete Stachylidium bicolor
Nagakubo et al. Phage tail-like nanostructures affect microbial interactions between Streptomyces and fungi
Alker et al. A modular plasmid toolkit applied in marine bacteria reveals functional insights during bacteria-stimulated metamorphosis
Li et al. PhcA and PhcR regulate ralsolamycin biosynthesis oppositely in Ralstonia solanacearum
CN111876514B (en) Rapid detection method for gibberellin miniascape generated in bakanae disease germ of rice
Hemmati et al. Identification of yeast and yeast-like symbionts associated with Hishimonus phycitis (Hemiptera: Cicadellidae), the insect vector of lime witches' broom phytoplasma
Jones et al. Ergot alkaloids contribute to the pathogenic potential of the fungus Aspergillus leporis
AU2021103864A4 (en) Engineering strain for producing 2-hydroxy-phenazine and use thereof
Ahearne et al. Chromosomal organization of biosynthetic gene clusters, including those of nine novel species, suggests plasticity of myxobacterial specialized metabolism

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