Construction method of high-yield siderophore pseudomonas fluorescens overexpression strain constructed based on ARTP mutagenesis

Abstract

The invention discloses a construction method of a high-yield siderophore pseudomonas fluorescens overexpression strain constructed based on ARTP mutagenesis, and the construction method of the strain comprises the following steps: (1) screening based on ARTP mutagenesis to obtain a siderophore synthesis positive regulation gene rpoS mutant gene sequence which is SEQ ID NO.1; and (2) constructing a pseudomonas fluorescens mutant strain with high siderophore yield. According to the obtained pseudomonas fluorescens overexpression strain, the siderophore yield reaches 89.8%. According to the invention, the siderophore is screened according to the gene mutation proportion to synthesize the positive regulation gene, and the screened rpoS mutant gene is overexpressed into the pseudomonas fluorescens, so that the RpoS protein in a high expression state promotes the synthesis and transport of the siderophore, and the high yield of the siderophore is realized.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for constructing a high-yield iron carrier fluorescing Pseudomonas overexpression strain based on ARTP mutagenesis. Background Art

[0002] ARTP mutagenesis technology is a new type of mutagenesis technology developed on the basis of plasma. This technology can produce a plasma jet with a temperature between 25-40°C and a high concentration of active particles under atmospheric pressure. This technology has the advantages of simple equipment, mild conditions, convenient operation and high mutation efficiency. It is an effective means of mutagenesis and breeding of high-yield active ingredient strains, and also provides a technical basis for the construction of high-yield active ingredient strains.

[0003] Iron is an essential element for the metabolism of living organisms and microorganisms. It is a cofactor or activator of many enzymes and participates in cellular transport and redox reactions. Although iron is abundant in the earth's crust, it mostly exists in an insoluble form in the natural environment. Organisms obtain Fe by secreting iron chelators - siderophores. 3+ , to meet the nutritional needs of iron. Iron carriers have many applications in food preservation, environmental governance and agriculture. They can reduce the utilization of iron ions by spoilage and pathogenic bacteria, delay food spoilage; chelate a variety of toxic metals and repair heavy metal pollution; reduce the nutritional utilization of plant pathogenic microorganisms and inhibit their growth; and make organic chelated iron fertilizers to alleviate plant iron deficiency symptoms.

[0004] At present, common synthesis methods of iron carriers include microbial fermentation, chemical synthesis, enzyme catalysis, etc., which generally have problems such as long cycle, high cost, low yield, and low purity. In addition, ARTP mutagenesis technology has not been used in the screening and construction of high-yield iron carrier strains, which limits the efficient preparation and application of iron carriers. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for constructing a high-yield iron carrier Pseudomonas fluorescens overexpression strain based on ARTP mutagenesis. The strain can be applied to industrial production on a large scale, and its iron carrier yield is as high as 89.8%.

[0006] The present invention proposes a method for constructing a high-yield siderophore Pseudomonas fluorescens overexpression strain based on ARTP mutagenesis, comprising the following steps:

[0007] (1) Based on ARTP mutagenesis screening, a mutant gene of the siderophore synthesis positive regulatory gene rpoS was obtained, and its sequence is SEQ ID NO.1.

[0008] (2) Construct a fluorescent Pseudomonas mutant strain with high iron carrier production.

[0009] Preferably, step (1) comprises mutagenizing Pseudomonas fluorescens using an ARTP mutagenesis breeding instrument, culturing the mutant sub-libraries of Pseudomonas fluorescens obtained by mutagenesis in medium with low iron ion concentration and medium with high iron ion concentration, respectively, extracting the whole genome for second-generation sequencing, and screening the mutant gene of the iron carrier synthesis positive regulatory gene rpoS according to the ratio of the total number of gene mutations.

[0010] Preferably, the ratio of the total number of gene mutations in step (1), that is, the total number of gene mutations in a low iron ion environment: the total number of gene mutations in a high iron ion environment, is not equal to 1.

[0011] Preferably, step (2) comprises cloning the rpoS mutant gene into the pBBR1MCS-2 vector in one step, transforming the recombinant vector into E. coli SM10 competent cells, screening to obtain positive donor cells, and conjugating the positive donor cells with the recipient cells Pseudomonas fluorescens at a ratio of 2:1 to obtain the Pseudomonas fluorescens strain PFRS.

[0012] Preferably, the amount of the rpoS mutant gene fragment used is 48-60 ng, and the amount of the vector pBBR1MCS-2 used is 100-110 ng.

[0013] Preferably, the bacterial concentration of the recipient cells and the donor cells is between OD60000.35-0.60, and the conjugation transfer time is 16-48h.

[0014] The fluorescent Pseudomonas overexpression strain obtained by the present invention has an iron carrier yield of up to 89.8%. The present invention uses ARTP mutagenesis technology to construct a fluorescent Pseudomonas mutant library, utilizes the difference in iron carrier synthesis regulatory genes of fluorescent Pseudomonas at different iron ion concentrations, screens iron carrier synthesis positive regulatory genes according to gene mutation ratios, and overexpresses the screened rpoS mutant gene into fluorescent Pseudomonas, so that the highly expressed RpoS protein promotes the synthesis and transport of iron carriers, thereby achieving high iron carrier yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the expression level of the wild-type Pseudomonas fluorescens strain and the Pseudomonas fluorescens PFRS gene pvdO and pvdE of the present invention.

[0016] Figure 2 It is the content of iron carrier produced by the wild-type fluorescing Pseudomonas strain and Pseudomonas fluorescens PFRS of the present invention. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the present invention. The change or adjustment of the relative relationship thereof shall also be regarded as the scope of the present invention without substantially changing the technical content.

[0018] In order to better understand the above technical scheme, the exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0019] The wild-type Pseudomonas fluorescens used in the following examples is Pseudomonas fluorescens PF08, which is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC M 2020065, address: Wuhan University, Wuhan, China, and a deposit date of July 17, 2019. Pseudomonas fluorescens PF08 was isolated and purified from corrupt large yellow croaker, a Gram-negative bacterium, cold-adapted and aerobic, with an optimal growth condition of Ph7.0, 28°C, and can be stably passaged.

[0020] Example 1: Screening and obtaining of positive regulatory mutation gene sequences for Pseudomonas fluorescens siderophore synthesis

[0021] 1. Cultivate the fluorescent Pseudomonas PF08 with the preservation number of CCTCC M 2020065 (this strain is the wild-type strain described below) to the end of the logarithmic phase, and adjust the bacterial solution concentration to OD600 of 0.65-0.80. Take 10 μL of bacterial solution and evenly spread it on the slide, and transfer it to the ARTP mutagenesis breeding instrument operation room. The distance between the slide and the plasma generator nozzle is about 2 mm, and the parameters are set: 120W, 10SLM, 15min. After the mutagenesis is completed, the fluorescent Pseudomonas mutant library attached to the slide is washed into a sterile solution, stored in a 10% glycerol test tube, frozen with liquid nitrogen, and stored at -80°C for later use. 2. Take the fluorescent Pseudomonas mutant library obtained in step 1 and culture it in a low-iron ion culture medium and a high-iron ion culture medium respectively until OD600 is 0.8-1.0, and use Beijing Quanshijin Bacteria Genomic DNA Kit was used to extract bacterial genomic DNA, and the extracted genomes were recorded as MLFe-DNA and MHFe-DNA, respectively.

[0022] 3. The MLFe-DNA and MHFe-DNA obtained in step 2 were respectively broken into fragments of about 450 bp in length and subjected to second-generation sequencing on the Illumina gene sequence analysis platform; the sequencing results were compared with the reference genes using BWA analysis software, and the sequencing depth and coverage relative to the wild-type genome of Pseudomonas fluorescens were calculated to obtain the mutation type and number of mutations of each gene in the mutant library genome; according to the principle that the total number of gene mutations in a low iron ion environment: the total number of gene mutations in a high iron ion environment is not equal to 1, the rpoS mutant gene sequence that is beneficial to the synthesis of iron carriers is obtained, and its sequence is as shown in SEQ ID NO.1.

[0023] Example 2: Construction of a high-yield iron carrier Pseudomonas fluorescens overexpression strain (Pseudomonas fluorescens PFRS)

[0024] 1. Use The rpoS mutant gene fragment synthesized by Shanghai Bioengineering was connected to the vector pBBR1MCS-2 using the MultiS one-step cloning kit. The reaction system was: 5×CE Multis buffer 4μL; vector 100-110ng; synthetic rpoS gene fragment 48-60ng; Exnase Multis 2μL; ddH2O was added to the total system to 20μL. The RpoS recombinant vector was obtained by reaction and stored at -20℃ for future use.

[0025] 2. The recombinant vector obtained in step 1 was transformed into E. coli SM10 competent cells, and a single clone was selected for colony PCR verification to obtain the donor strain. The donor strain and the recipient strain, Pseudomonas fluorescens, were cultured to OD600 of 0.45, and conjugated at a ratio of 2:1. The successfully expressed strains were screened using kanamycin sulfate (Km), and a single clone was selected for PCR and sequencing verification to obtain a high-yield iron carrier Pseudomonas fluorescens RpoS overexpression strain, Pseudomonas fluorescens PFRS.

[0026] Example 3: Verification of high-yield iron carrier capability

[0027] 1. The RpoS overexpression strain of Pseudomonas fluorescens obtained in Example 2 and the wild-type Pseudomonas fluorescens were inoculated into MM9 culture medium at a bacterial liquid volume ratio of 1:100, and cultured at 28°C and 200 rpm for 24 h. Total RNA was extracted with reagents, and genomic DNA was removed using DNase I. RT-PCR was used to detect the expression of iron carrier synthesis and transport genes pvdO and pvdE. Figure 1 shown.

[0028] 2. Centrifuge the bacterial culture in step 1, take 3 mL of the supernatant and add an equal volume of CAS solution, mix well and place in the dark for 1 hour, measure the OD value at a wavelength of 630 nm, and calculate the siderophore production. Figure 2 shown.

[0029] The results are as follows Figure 1 and 2 As shown in the results, the expression levels of pvdO and pvdE and the iron carrier production of the RpoS overexpression strain of Pseudomonas fluorescens were significantly higher than those of the wild-type Pseudomonas fluorescens (P < 0.05), and the iron carrier production of the RpoS overexpression strain of Pseudomonas fluorescens was as high as 89.8%, while the iron carrier production of the wild-type Pseudomonas fluorescens was 66.9%. Pseudomonas fluorescens regulates the synthesis of iron carriers and the expression of transport proteins through the overexpression of RpoS protein, thereby promoting the production of iron carriers.

[0030] In summary, the present invention clones the iron carrier positive regulatory protein RpoS gene into Pseudomonas fluorescens, and through high expression of RpoS protein, effectively promotes the synthesis and transport of Pseudomonas fluorescens iron carriers, thereby achieving high production of iron carriers.

[0031] The RpoS overexpression strain of Pseudomonas fluorescens is a Gram-negative bacterium, cold-adapted and aerobic, and can be stably subcultured. The LB medium has the following culture conditions: pH 7.0, temperature 28°C, and kanamycin sulfate 50 μg / ml.

[0032] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0033] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

[0034] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the present invention. The change or adjustment of the relative relationship thereof shall also be regarded as the scope of the present invention without substantially changing the technical content.

Claims

1. A method for constructing a high-yield siderophore Pseudomonas fluorescens overexpression strain based on ARTP mutagenesis, comprising the following steps: (1) Based on ARTP mutagenesis screening, the mutant gene sequence of the siderophore synthesis positive regulatory gene rpoS was obtained, and its sequence is SEQ ID NO.1; (2) Construct a fluorescent Pseudomonas mutant strain with high iron carrier production.

2. The construction method according to claim 1, characterized in that: Step (1) includes using an ARTP mutagenesis breeding instrument to perform mutagenesis on Pseudomonas fluorescens, culturing the mutant sub-libraries of Pseudomonas fluorescens obtained by mutagenesis in low iron ion and high iron ion concentration culture media, respectively, extracting the whole genome for second-generation sequencing, and screening the sequence of the mutant gene of the iron carrier synthesis positive regulatory gene rpoS according to the ratio of the total number of gene mutations.

3. The construction method according to claim 2, characterized in that: The ratio of the total number of gene mutations in step (1), that is, the total number of gene mutations in a low iron ion environment: the total number of gene mutations in a high iron ion environment, is not equal to 1.

4. The construction method according to claim 1, characterized in that: Step (2) includes cloning the rpoS mutant gene into the pBBR1MCS-2 vector in one step, transforming the recombinant vector into E. coli SM10 competent cells, screening to obtain positive donor cells, and conjugating the positive donor cells with the recipient cells Pseudomonas fluorescens at a ratio of 2:1 to screen to obtain the Pseudomonas fluorescens strain PFRS.

5. The construction method according to claim 4, characterized in that: The amount of rpoS mutant gene fragment used is 48-60 ng, and the amount of vector pBBR1MCS-2 used is 100-110 ng.

6. The construction method according to claim 5, characterized in that: The bacterial concentration of the recipient cells and donor cells is between OD600 0.35-0.60, and the conjugation transfer time is 16-48h.

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