AU2021103349A4

AU2021103349A4 - Pseudomonas Chlororaphis QOHPHZ-8 for producing 1-Hydroxy-Phenazine (1-OH-PHZ) and use thereof

Info

Publication number: AU2021103349A4
Application number: AU2021103349A
Authority: AU
Inventors: Ling Li; Piwu Li; Kaiquan Liu; Ruiming WANG; Tengfei WANG
Original assignee: Qilu University of Technology
Current assignee: Qilu University of Technology
Priority date: 2020-09-25
Filing date: 2021-06-15
Publication date: 2021-08-05
Anticipated expiration: 2029-06-15
Also published as: CN112094795B; ZA202107098B; CN112094795A

Abstract

The present disclosure particularly relates to a Pseudomonas chlororaphis QOHPHZ-8 for producing 1-hydroxy-phenazine (1-OH-PHZ) and use of the Pseudomonas chlororaphis QOHPHZ 8. The present disclosure screens and obtains a strain of Pseudomonas chlororaphis Qlu-1. The strain has a typical gene cluster for synthesis of a phenazine compound, but cannot naturally synthesize and secrete the 1-OH-PHZ. The present disclosure first prepares QOHPHZ-1 by replacing a phzO gene with a phzS gene and obtains an accumulation of the 1-OH-PHZ. In order to further obtain a high-yielding strain of the 1-OH-PHZ, the present disclosure obtains QOHPHZ-8 strain by implementing subsequent optimization of the QOHPHZ-1 strain. The yield of the 1-OH PHZ per unit time is increased by 28 times, and the QOHPHZ-8 strain is expected to be an engineering strain used in the fields of agricultural production and algae control. 1/3 A B 1 2 3 4 5 1 2 3 FIG. 1 FIG. 2

Description

1/3

A B 1 2 3 4 5 1 2 3

FIG. 1

FIG. 2

PSEUDOMONAS CHLORORAPHIS QOHPHZ-8 FOR PRODUCING 1-HYDROXY PHENAZINE (1-OH-PHZ) AND USE THEREOF

TECHNICAL FIELD

[0001] The present disclosure belongs to the technical field of microorganism engineering strains and particularly relates to a Pseudomonas chlororaphis QOHPHZ-8 for producing 1-hydroxy-phenazine (1-OH-PHZ) and use thereof as an engineering strain.

BACKGROUND

[0002] Information for disclosing this background section is only for the purpose of increasing understanding of the general background of the present disclosure, and is not necessarily regarded as an acknowledgement or any form of suggestion that the information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Pesticides are important for agricultural production and are also of great significance for maintaining agricultural ecological environment. Biological pesticides are safe for non-target organisms, have less toxic and side effects, and are environmentally friendly. Compared with chemical pesticides, biological pesticides have many unique advantages and promising development prospects. At the same time, biological pesticides also face development bottlenecks, such as a low fermentation titer of biocontrol for Pseudomonas spp.

[0004] Phenazines are nitrogen-containing heterocyclic pigments. Current research has found that almost all the phenazines exhibit biocontrol activity against bacteria and fungi. Among the Pseudomonas spp, main antibiotics include phenazine-1-carboxylic acid (PCA), 1 hydroxy-phenazine (1-OH-PHZ), 2-hydroxy-phenazine (2-OH-PHZ), pyocyanin (PYO), phenazine-1-carboxamide (PCN), etc.

[0005] The 1-OH-PHZ is a secondary metabolite of the Pseudomonas spp and causes cell death by increasing oxygen free radicals in cells. The substance has optimal stability and is expected to exert stable anti-microorganism and disease-preventing effects when applied as a microbial pesticide. In addition, studies have shown that the 1-OH-PHZ also has a desirable inhibitory effect on Aspergillus terreus, green algae and cyanobacteria, and is also expected to be used in marine pollution control. Currently, methods of synthesisof the 1-OH-PHZ are mainly through biosynthetic pathways and chemical synthetic pathways. According to literature reports, the chemical synthetic pathways of the 1-OH-PHZ include: (1) nitrogen oxide of the 1 OH-PHZ is obtained from a cycloaddition reaction of benzofuran oxide and cyclohexanedione, and the 1-OH-PHZ is prepared by reducing a nitrogen oxidation part through sodium hydrosulfite; (2) Haddadin et al. also proposed to react the benzofuran oxide with 1,2 cyclohexanedione under triethylamine and nitrogen for 7-8 hours to obtain a dark brown reaction mixture, acidify the mixture to obtain a mixture of1-hydroxy-phenazine nitrogen oxide and obtain the 1-OH-PHZ after treatment with sodium hydrosulfite. In the biosynthetic pathways, current studies show that a synthesis of the 1-OH-PHZ is mainly related to a shikimic acid pathway in Pseudomonas spp. Shikimic acid is converted to chorismic acid, and phenazine-1-carboxylic acid is further synthesized and finally converted into the 1-OH-PHZ under an action of PhzS protein. Based on advantages of the biosynthetic pathways, it is of high research value to obtain an engineering strain with a higher yield by modifying the Pseudomonas strain.

[0006] Based on previous study results of inventors, Pseudomonas chlororaphiscontains a core gene component for synthesis of phenazines and related modifying genes for phenazines. Besides, Pseudomonas chlororaphis itself has low pathogenicity and is safe to use. Since chemical synthesis conditions of the 1-OH-PHZ are harsh and unfriendly to environment, it is of great significance to obtain an engineering strain with a high yield of the 1-OH-PHZ based on microorganisms.

SUMMARY

[0007] Based on the above background art, the present disclosure first obtains a strain of Pseudomonas chlororaphis Qlu-1 through screening. The strain has a typical gene cluster phzABCBEFG for phenazine synthesis and can also produce phenazine-1-carboxylic acid (PCA) and 2-hydroxy-phenazine (2-OH-PHZ). However, in its natural form Pseudomonas chlororaphis Qlu-1 cannot synthesize the 1-hydroxy-phenazine (1-OH-PHZ). The present disclosure first replaces a phenazine modifying gene phzO in the Qlu-1 with a phenazine modification gene phzS to obtain Pseudomonas chlororaphis QOHPHZ-1, such that the strain can express 1-OH-PHZ. The maximum yield of the 1-OH-PHZ of the Pseudomonas chlororaphis QOHPHZ-1 is about 20 mg/L, which is far from meeting use requirements. Further, Pseudomonas chlororaphis QOHPHZ-8 is obtained by gene modification of the QOHPHZ-1, has the yield of the 1-OH-PHZ reaching about 560 mg/L, which is increased by 28 times compared with that of the 1-OH-PHZ produced by the QOHPHZ-1, and is expected to be an engineering strain used in the fields of agricultural production and algae control.

[0008] Based on the above technical effect, the present disclosure provides the following technical solution:

[0009] In a first aspect of the present invention, there is provided a Pseudomonas chlororaphis QOHPHZ-1 for producing 1-hydroxy-phenazine (1-OH-PHZ), wherein the Pseudomonas chlororaphis QOHPHZ-1 is obtained using a Pseudomonas chlororaphis Qlu-1 as a starting strain and by replacing a phzO gene in the Qlu-1 with exogenous phzS; with the starting strain Pseudomonas chlororaphis Qlu-1 being preserved in the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China on May 8, 2020, and having a biological accession number of CCTCC NO: M2020108.

[00010] Based on the method described in the first aspect, the Pseudomonas chlororaphis Qlu-1 is now capable of expressng the 1-OH-PHZ after being modified, for the first time. The present invention carried out a subsequent optimization of the strain to obtain a strain Pseudomonas chlororaphisQOHPHZ-8 capable of high-yielding 1-OH-PHZ.

[00011] In a second aspect of the present invention, a Pseudomonas chlororaphis QOHPHZ-8 producing 1-OH-PHZ is provided, wherein the Pseudomonas chlororaphis QOHPHZ-8 uses the Pseudomonas chlororaphis QOHPHZ-1 of the first aspect as a starting strain, has copies of an insertion of a tktA-ppsA-phzC-aroB-aroD-aroEgene fragment genome, and lacks genes pykF, Ion, psrA, parS, rsmE, and rpeA.

[00012] In a third aspect of the present invention, there is provided use of the Pseudomonas chlororaphis QOHPHZ-1 of the first aspect and/or the Pseudomonas chlororaphis QOHPHZ-8 of the second aspect as an engineering strain for producing 1-OH PHZ.

[00013] In a fourth aspect of the present invention, a bacterial agent is provided and includes one or a mixed strain of the Pseudomonas chlororaphis QOHPHZ-1 of the first aspect and the Pseudomonas chlororaphisQOHPHZ-8 of the second aspect, or a culture thereof.

[00014] In a fifth aspect of the present invention, there is provided use of the bacterial agent of the forth aspect in preparing an agricultural product.

[00015] In a sixth aspect of the present invention, there is provided an algae control preparation which may include the bacterial agent of the fourth aspect.

[00016] One or more of the above technical solutions have the following beneficial effects:

[00017] 1. The Pseudomonas chlororaphis has lower pathogenicity and is safer than existing Pseudomonas aeruginosa as an engineering strain. With regard to the defect that the fermentation capacity of the current biosynthetic pathways is insufficient, the optimization method of the present invention significantly improves the yield of the modified strain.

[00018] 2. The research on synthetic pathways of phenazines in Pseudomonas chlororaphisin the field shows that the same target is usually related to multiple physiological activities and when multiple targets are changed at the same time, the result may be unexpected. The gene editing method provided by the present disclosure effectively improves the yield of the 1-OH-PHZ and has reference significance for further clarifying a synthesis mechanism of phenazine substances in microorganisms.

BRIEF DESCRIPTION OF DRAWINGS

[00019] The accompanying drawings of the specification which constitute a part of the present invention provide further understanding of the present invention. The schematic examples of the present invention and description thereof are intended to explain the present invention and are not intended to constitute an improper limitation of the present invention.

[00020] FIG.1 shows upstream and downstream amplification of a psrA gene in Example 2;

[00021] FIG. 1(A), 1 is a DNA Ladder, 2-3 are an upstream amplified sequence of the psrA, and 4-5 are a downstream amplified sequence of the psrA; and

[00022] FIG. 1(B), 1 is a DNA Ladder and 2-3 are an upstream and downstream fusion fragment of thepsrA.

[00023] FIG. 2 shows a biparental hybrid double-resistance plate screening result of a psrA gene in Example 2.

[00024] FIG. 3 shows screening of psrA gene double-exchange positive monoclones by photocopying in Example 2.

[00025] FIG. 4 shows a PCR verification of a psrA-knockout strain in Example 2;

[00026] Detection of external primers: 1 is a psrA-knockout strain genome as template amplified fragment; 2 is DNA Ladder; 3 is blank control; and 4 is a wild strain genome as template amplified fragment;

[00027] Detection of internal primers: 1 is a wild strain genome as template amplified fragment; 2 is DNA Ladder; 3 is psrA-knockout strain genome as template amplified fragment; and 4 is blank control.

[00028] FIG. 5 is a diagram of yield of -OH-PHZ in different strains.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[00029] It should be noted that the following detailed description is exemplary and aims to further describe the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by a person of ordinary skill in the art to which the present disclosure belongs.

[00030] It should be noted that the terms used herein are merely used for describing the specific embodiments, but are not intended to limit exemplary embodiments of the present invention. As used herein, a singular form is intended to include a plural form unless otherwise indicated obviously in the context. Further, it should be further understood that the terms "includes" and/or "including" used in this specification specify the presence of features, steps, operations, devices, components and/or of combinations thereof.

[00031] As described in the background art, in order to solve the above technical problem that the yield of the 1-OH-PHZ through biosynthetic pathways is not high, the present invention provides a Pseudomonas chlororaphisQOHPHZ-8 capable of high-yielding 1-OH-PHZ.

[00032] In a first aspect of the present invention, there is provided a Pseudomonas chlororaphis QOHPHZ-1 for producing 1-hydroxy-phenazine (1-OH-PHZ), wherein the Pseudomonas chlororaphis QOHPHZ-1 uses a Pseudomonas chlororaphis Qlu-1 as a starting strain and is obtained by using an exogenous phzS gene to replace a phzO gene in the Qlu-1; and the starting strain Pseudomonas chlororaphis Qlu-1 is preserved in the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China on May 8, 2020, and has a biological accession number of CCTCC NO: M2020108.

[00033] Related gene fragments in the Pseudomonaschlororaphis provided by the first to second aspects of the present invention can all be edited by gene editing methods commonly used in the field, and a method of knocking out a gene phzO or introducing an exogenous phzS gene includes but is not limited to gene editing through a siRNA gene silencing mechanism, a zinc finger nuclease (ZFNs), a transcription activator-like effector nuclease (TALENS), a CRISPER-Cas9 system, a lentiviral vector or a plasmid transfection.

[00034] Further, preferably, the gene knockout or introduction may be achieved by the plasmid transfection in a specific embodiment of the present invention.

[00035] In a second aspect of the present invention, there is provided a Pseudomonas chlororaphis QOHPHZ-8 producing 1-OH-PHZ, wherein the Pseudomonas chlororaphis QOHPHZ-8 uses the Pseudomonas chlororaphis QOHPHZ-1 of the first aspect as a starting strain, has copies of an insertion of a tktA-ppsA-phzC-aroB-aroD-aroEgene fragment genome, and lacks genes pykF, Ion, psrA, parS, rsmE, and rpeA.

[00036] In a third aspect of the present invention, Pseudomonas chlororaphisQOHPHZ-1 of the first aspect and/or the Pseudomonas chlororaphis QOHPHZ-8 of the second aspect are used as an engineering strains for producing 1-OH-PHZ.

[00037] In a fourth aspect of the present invention, there is provided a bacterial agent which includes one or a mixed strain of the Pseudomonas chlororaphis QOHPHZ-1 of the first aspect and the Pseudomonaschlororaphis QOHPHZ-8 of the second aspect, or a culture thereof.

[00038] Preferably, the culture may include but is not limited to a fermentation product of the strain, or a metabolite isolated after a fermentation of the strain.

[00039] Preferably, a dosage form of the bacterial agent may include but is not limited to liquid, powder, a granule or a tablet.

[00040] Further, preferably a dosage form of the bacterial agent may be wettable powder, a water dispersible granule and an aqueous suspension.

[00041] Preferably, the bacterial agent may also include a necessary auxiliary material.

[00042] In a fifth aspect of the present invention, there is provided use of the bacterial agent of the forth aspect in preparing an agricultural product.

[00043] Preferably, the agricultural product may include but is not limited to a pesticide, a fertilizer and a culture medium.

[00044] Further, preferably the pesticide is prepared from the bacterial agent of the fifth aspect as an active ingredient, or applied in combination with other pesticides, microbial pesticides, etc.

[00045] Further, preferably the fertilizer may include but be not limited to an ammonium phosphate fertilizer, a macro-element water-soluble fertilizer, a medium-element fertilizer, a biological fertilizer, an organic fertilizer, or a multi-dimensional field energy concentrated organic fertilizer.

[00046] Further, preferably, the culture medium may be a greenhouse seedling culture medium.

[00047] In the sixth aspect of the present invention, an algae control preparation is provided and includes the bacterial agent of the fourth aspect.

[00048] The algae control preparation may be applied to, but be not limited to ecological wetland treatment, sewage treatment, removal of algae in breeding industry, and marine pollution treatment. Existing studies have confirmed that the 1-OH-PHZ also has a good inhibitory effect on some algae and better environmental compatibility than a traditional chemical reagent as an environmental treatment reagent.

[00049] In order to enable those skilled in the art to more clearly understand the technical solutions of the present invention, the technical solutions of the present invention will be described in detail below with reference to specific examples. Unless otherwise specified, the related reagents in the following examples are all commercially available products.

Example 1

[00050] In this example, a method for preparing Pseudomonas chlororaphis QOHPHZ-1 based on Pseudomonas chlororaphis Qlu-1 was provided and the preparation method included the following steps:

1. Pseudomonas chlororaphis Qlu-1 was inoculated into a KB(A*) culture medium, shaking culture was conducted overnight at 30°C with a shaker at 180 rpm, a genome of the Qlu-1 was extracted with a genome extraction kit, and the extracted Qlu-1 genome was stored at 20°C for later use.

2. A phzO gene and its upstream and downstream sequences were searched in sequenced Qlu-1 genome data, the Qlu-1 strain genome was used as a template, and phzO-F1/phzO-R1 and phzO-F2/phzO-R2 were respectively used as primers to amplify an upstream fragment phzO-U and a downstream fragment phzO-D of the phzO gene; and the phzO-U, phzO-D and phzO-F1/phzO-R2 were used as templates to amplify a phzO upstream and downstream fusion fragment phzO-UD. The fusion fragment phzO-UD was ligated with a knockout plasmid pkl8moBsacB to construct a recombinant plasmid pkl8-phzO-UD through restriction digestion.

[00051] The pkl8-phzO-UD was introduced into Escherichiacoli S17-1 (X) by heat shock transformation, the Escherichia coli S17-1 (X) and the Pseudomonas chlororaphis Qlu-1 were subjected to a biparental hybrid culture, such that the recombinant plasmid pkl8-phzO-UD was introduced into the Pseudomonas chlororaphis Qlu-1. After the Pseudomonas chlororaphis Qlu-1 and Escherichiacoli S17-1 (X) were co-cultured, the co-cultured bacteria were spread on a KB(A*K) double antibody plate, and single colonies were selected. A phzO-knockout Qlu-1 strain was jointly screened by sucrose plate screening, photocopy screening and PCR screening. After fermentation, HPLC detection showed that only phenazine-1-carboxylic acid accumulated in a fermentation broth, and the strain was named QPCA.

[00052] The Qlu-1 genome was used as template, the phzO-F1/phzO-R1-2 and phzO-F2 2/phzO-R2 were used as primers to amplify an upstream fragment phzO-U2 and a downstream fragment phzO-D2 of the phzO gene, a synthesized phzS gene was used as a template, phzS F1/phzS-R1 were used as primers to amplify a fragment phzS-2, and phzO-U2, phzO-D2, phzS 2 and phzO-F1/phzO-R2 were used as templates to amplify a phzS-introduced fragment phzS IN that fused the upstream and downstream of the phzO gene. The fusion fragment phzS-IN was ligated with the knockout plasmid pkl8moBsacB to construct a recombinant plasmid pkl8 phzS-IN through restriction digestion. The recombinant plasmid pkl8-phzS-IN was introduced into Escherichia coli S17-1 (X) by heat shock transformation. The Escherichia coli S17-1 (X) and the Pseudomonas chlororaphis Qlu-1 were subjected to a biparental hybrid culture, such that the recombinant plasmid pkl8-phzS-IN was introduced into the Pseudomonas chlororaphis QPCA.

[00053] A phzS inserted Qlu-1 strain was jointly screened by sucrose plate screening, photocopy screening and PCR screening. After fermentation, HPLC detection showed that after 48 h, only 1-OH-PHZ accumulated in a fermentation broth, and the strain was named QOHPHZ-1. After fermentation with the QOHPHZ-1, an extraction was conducted by ethyl acetate. HPLC detection found that the QOHPHZ-1 could produce 20.4 mg/L of the1-OH-PHZ at 96 h.

Example 2

[00054] In this example, a method for preparing Pseudomonas chlororaphis QOHPHZ-8 based on Pseudomonas chlororaphis QOHPHZ-1 in example 1 was provided and the preparation method included the following steps:

[00055] Based on the QOHPHZ-1, phenazine synthesis leading pathway-related genes phzC, aroB, aroD and aroE and central metabolism-related genes tktA andppsA were amplified respectively. A Bglbric modular plasmid pBbB8k-GFP was used as a medium plasmid, a single modular plasmid was constructed separately, and the genes were integrated into tandem gene pBbB8K-tktA-ppsA-phzC-aroB-aroD-aroE by using a modular principle.

[00056] The tandem gene was excised from the pBbB8K-tktA-ppsA-phzC-aroB-aroD aroE, ligated to an integration plasmid pUC18-mini-Tn7T-Gm, and integrated into a QOHPHZ 1 genome with a helper plasmid pTNS2 through the pUC18-mini-Tn7T-Gm plasmid to obtain Pseudomonas chlororaphis QOHPHZ-2, and after fermentation, it was found that the yield of the 1-OH-PHZ in the strain QOHPHZ-2 reached 83.8 mg/L within 96 h.

[00057] The Pseudomonas chlororaphis QOHPHZ-2 was used as a starting strain and pykF, Ion, psrA, parS, rsmE and rpeA genes were knocked out.

[00058] A seamless knockout of a psrA gene was used as an example to introduce gene manipulation. A kit was used to extract a Qlu-1 genome, the psrA gene and its upstream and downstream sequences in a sequenced Qlu-1 genome sequence were searched, and a PCR was used to catch and connect an upstream and downstream fragment psrA-ud of the psrA gene.

[00059] The psrA-ud fragment was connected to a plasmid pkl8moBsacB by a seamless cloning technology to construct a psrA knockout plasmid pK18-psrA-ud. The pK18- psrA-ud was introduced into Escherichiacoli S17-1 (X) by heat shock transformation.

[00060] After Pseudomonas chlororaphis QOHPHZ-4 and the Escherichia coli S17-1 (X) were co-cultured, the co-cultured bacteria were spread on a KB (AK+) double antibody plate, and single colonies were selected. The single colonies were spread on a 15% sucrose KB plate containing ampicillin, and the QOHPHZ-4 knockout psrA strain was screened and verified by PCR.

[00061] After the negative regulatory genes pykF, Ion, psrA, parS, rsmE and rpeA were successively knocked out in a QOHPHZ-2 genome by the same method, a QOHPHZ-8 strain capable of high-yielding 1-OH-PHZ was obtained. After fermentation, an extraction was conducted by ethyl acetate. HPLC detection found that the QOHPHZ-8 could produce 560 mg/L of the 1-OH-PHZ at 96 h.

[00062] In FIGS. 1-4, a psrA knockout was used as an example to illustrate the above steps. In FIG. 5, the pykF, lon, psrA, parS, rsmE and rpeA genes in the Pseudomonas chlororaphis QOHPHZ-2 were knocked out in sequence and the knockout Pseudomonas chlororaphis QOHPHZ-2 were named as QOHPHZ-3, QOHPHZ-4, QOHPHZ-5, QOHPHZ-6, QOHPHZ-7 and QOHPHZ-8. The yields of the 1-OH-PHZ of the QOHPHZ-3, QOHPHZ-4, QOHPHZ-5, QOHPHZ-6, QOHPHZ-7 and QOHPHZ-8 strains were shown in FIG. 5.

Comparative Example 1

[00063] In this example, a method of expressing related genes by a plasmid expression is provided. First, the genes were respectively constructed into a pBbB8K-GFP plasmid, a single gene plasmid was integrated into tandem gene pBbB8K-tktA-ppsA-phzC-aroB-aroD-aroE through Bglbric modular plasmid operation rules, and the recombinant plasmid was introduced into a strain QOHPHZ-1 by electroporation transformation to obtain a strain QOHPHZ-2-2. In KB additionally added with Kan antibiotics, the 1-OH-PHZ was also produced in the QOHPHZ 2-2 through fermentation and had the yield of 52.6 mg/L. Compared with the plasmid expression, a genome integration expression is stable and more efficient. (The yield of the 1 OH-PHZ of the tktA-ppsA-phzC-aroB-aroD-aroE genome integrated strain QOHPHZ-2 was 83.8 mg/L).

[00064] The foregoing is merely illustrative of the preferred examples of the present disclosure and is not intended to limit the present disclosure, and various changes and modifications may be made by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and scope of the present disclosure should be included within the protection scope of the present disclosure.

[00065] Any discussion of documents, devices, acts or knowledge in this specification is included to explain the context of the invention. It should not be taken as an admission that any of the material forms a part of the prior art base or the common general knowledge in the relevant art in Australia on or before the priority date of the disclosure herein.

[00066] Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other features, integers, steps, components to be grouped therewith.

Claims

CLAIMS:

1. A Pseudomonas chlororaphisQOHPHZ-1 for producing 1-hydroxy-phenazine (1-OH-PHZ), wherein the Pseudomonas chlororaphis QOHPHZ-1 is produced by using a Pseudomonas chlororaphis Qlu-1 as a starting strain and Pseudomonas chlororaphis QOHPHZ-1 is obtained by replacing a phzO gene in the Qlu-lusing an exogenous phzS gene ; and the starting strain Pseudomonas chlororaphis Qlu-1 is preserved in the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China on May 8, 2020, and has a biological accession number of CCTCC NO: M2020108.

2. The Pseudomonas chlororaphis QOHPHZ-1 for producing 1-hydroxy-phenazine according to claim 1, wherein a method of knocking out the gene phzO or introducing the exogenous phzS gene comprises gene editing through a siRNA gene silencing mechanism, a zinc finger nuclease, a transcription activator-like effector nuclease, a CRISPER-Cas9 system, a lentiviral vector or a plasmid transfection; and preferably, the gene knockout or introduction is achieved by the plasmid transfection.

3. A Pseudomonas chlororaphisQOHPHZ-8 for producing 1-hydroxy-phenazine (1-OH-PHZ), wherein the Pseudomonas chlororaphis QOHPHZ-8 uses the Pseudomonas chlororaphis QOHPHZ-1 according to claim 1 or claim 2 as a starting strain, has multiple copies of an insertion of a tktA-ppsA-phzC-aroB-aroD-aroE gene fragment genome, and lacks genes pykF, Ion, psrA, parS, rsmE, and rpeA.

4. A bacterial agent, comprising one or a mixed strain of the Pseudomonas chlororaphis QOHPHZ-1 according to claim 1 or claim 2 and the Pseudomonas chlororaphis QOHPHZ-8 according to claim 3, or a culture thereof;

wherein the culture comprises a fermentation product of the strain, or a metabolite isolated after a fermentation of the strain;

or, a dosage form of the bacterial agent comprising liquid, powder, a granule or a tablet; further preferably, a dosage form of the bacterial agent comprises wettable powder, a water dispersible granule, or an aqueous suspension;

or, the bacterial agent also comprises a necessary auxiliary material.

5. An algae control preparation, comprising the bacterial agent according to claim 4.