Construction method of pseudomonas fluorescens with high siderophore yield

Abstract

The invention discloses a construction method of pseudomonas fluorescens with high siderophore yield. The construction method comprises the following steps: S1, preparing a CsrA succinylated gene amplified fragment; s2, constructing a CsrA succinylated recombinant vector; and S3, constructing the pseudomonas fluorescens with high siderophore yield. The CsrA protein in the desuccinylation state is cloned into the pseudomonas fluorescens, so that the CsrA protein in the desuccinylation state is at a high expression level, the synthesis of the siderophore and the expression of transport genes pvdA and pvdE are further promoted, the high yield of the siderophore is realized, and the yield is up to 89.4%.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for constructing a high-yield iron carrier fluorescent pseudomonas. Background Art

[0002] Iron is an essential element for the growth and metabolism of many organisms, but in the natural environment, the direct availability of iron to microorganisms is usually low. Iron carriers are low molecular weight substances that mainly exist in microorganisms and can bind trivalent iron ions and supply microbial cells, making iron more easily absorbed and utilized by organisms, thus playing an important role in the survival and adaptation of microorganisms to the environment. It is also closely related to the nutrient absorption and health of plants.

[0003] However, not all microorganisms can produce siderophores. Whether a microorganism produces siderophores depends on its genetic characteristics, living environment, and demand for iron. Even among microorganisms that produce siderophores, the type and amount of siderophores produced may vary. Different microbial species may produce different types of siderophores, which may differ in structure and properties, thus affecting their ability to bind to iron ions and their biological functions. Therefore, screening and constructing more strains with high siderophore production capacity is of great significance for enriching the resources of siderophore-producing strains and developing new antibacterial and biocontrol agents. Summary of the invention

[0004] The purpose of the present invention is to provide a method for constructing a high-yield iron carrier fluorescent Pseudomonas.

[0005] The present invention provides a method for constructing a high-yield iron carrier Pseudomonas fluorescens, comprising the following steps:

[0006] S1: preparing a CsrA desuccinylation gene amplified fragment, the sequence of which is SEQ ID NO.1;

[0007] S2: Construction of CsrA desuccinylation recombinant vector;

[0008] S3: Construction of fluorescent Pseudomonas with high iron carrier production.

[0009] Preferably, the step of preparing the CsrA desuccinylation gene amplification fragment includes extracting the whole genome DNA of Pseudomonas fluorescens as an amplification template; using CsrA (K38R) -F and CsrA (K38R) -R as amplification primers and using a high-fidelity enzyme for PCR amplification.

[0010] Preferably, the sequence of primer CsrA(K38R)-F (SEQ ID NO.2) is

[0011] 5'-GCATCAATGCTCCCAGAGATGTTGCCGTGCAT-3'; the sequence of primer CsrA(K38R)-R (SEQ ID NO. 3) is 5'-TCTGGGAGCATTGATGCCGATTCTTACTTGCT-3'.

[0012] Preferably, the step of constructing the CsrA desuccinylation recombinant vector comprises purifying the amplified fragment of the CsrA desuccinylation gene and cloning and recombining it into the vector pBBR1MCS-2 in one step.

[0013] Preferably, the step of constructing a high-yield iron carrier-producing Pseudomonas fluorescens CsrA desuccinylation transformant comprises transforming the CsrA desuccinylation recombinant vector into donor cells E. coli SM10, and screening to obtain positive donor cells; and conjugating the donor cells E. coli SM10 with recipient cells Pseudomonas fluorescens, and screening to obtain high-yield iron carrier-producing Pseudomonas fluorescens.

[0014] Preferably, the amount of the CsrA desuccinylation gene amplified and purified fragment used is 10-15 ng, and the amount of the vector pBBR1MCS-2 used is 100-110 ng.

[0015] Preferably, the conjugation ratio of recipient cells to positive donor cells is 1:2-1:3.5, and the conjugation transfer time is 16-36 hours.

[0016] Preferably, the siderophore produced by the high-yield siderophore-producing Pseudomonas fluorescens is a hydroxamic acid-producing siderophore.

[0017] According to a method for constructing a high-yield iron carrier fluorescent Pseudomonas strain in an embodiment of the present invention, the present invention clones a desuccinylated CsrA protein into the fluorescent Pseudomonas, so that the desuccinylated CsrA protein is at a high expression level, thereby promoting the expression of iron carrier synthesis and transport genes pvdA and pvdE, and achieving high iron carrier production, with a yield of up to 89.4%. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the expression level of genes pvdA and pvdE of the wild-type fluorescent Pseudomonas strain and the CsrA desuccinylation transformant strain of the present invention.

[0019] Figure 2 It is the content of iron carrier produced by the wild-type fluorescent Pseudomonas strain and the CsrA desuccinylated transformant strain of the present invention. DETAILED DESCRIPTION

[0020] 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.

[0021] 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.

[0022] The fluorescing Pseudomonas used to construct a high-yield iron carrier 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 M2020065, address: Wuhan University, Wuhan, China, and a deposit date of July 17, 2019.

[0023] Pseudomonas fluorescens PF08 was isolated and purified from corrupted large yellow croaker. It is a Gram-negative bacterium that is cold-adaptive and aerobic. Its optimal growth conditions are: Ph7.0, 28℃, and it can be stably subcultured.

[0024] Example 1: Screening of high-yield regulatory proteins for Pseudomonas fluorescens siderophore

[0025] 1. Pseudomonas fluorescens PF08 was cultured in high iron ion concentration and low iron ion concentration culture media to the mid-to-late logarithmic period, and the total protein of Pseudomonas fluorescens in the two environments was extracted using a bacterial total protein extraction kit, and the protein content was determined using a BCA kit.

[0026] 2. Take the bacterial whole protein extracted in step 1, add trypsin solution at a ratio of 1:50 (protease: protein, m / m), enzymatically hydrolyze overnight to obtain peptides, and then transfer to succinylated resin for incubation, elution, and vacuum drying to obtain succinylated modified enriched peptides.

[0027] 3. Use liquid chromatography-mass spectrometry to perform proteomic and succinylation modification group analysis on the peptides obtained in step 2, with P≤0.05 and a differential expression change of >1.5 as the threshold for significant upregulation and <1 / 1.5 as the threshold for significant downregulation, to screen the differentially modified proteins of Pseudomonas fluorescens under high iron ion and low iron ion concentration environments, and obtain the desuccinylated CsrA protein, whose succinylation modification site is at lysine at amino acid position 38 (Lys-38).

[0028] Example 2: Construction of a mutant strain of Pseudomonas fluorescens that produces high iron carriers

[0029] 1. The Lys-38 of the CsrA protein of Pseudomonas fluorescens was mutated to Arg (K to R) by point mutation technology to retain the positive charge and simulate the desuccinylated lysine. The whole genome DNA of Pseudomonas fluorescens was extracted and used as an amplification template. The point mutation primers CsrA (K38R)-F (SEQ ID NO.2): 5'-GCATCAATGCTCCCAGAGATGTTGCCGTGCAT-3' and CsrA (K38R)-R (SEQ ID NO.3): 5'-TCTGGGAGCATTGATGCCGATTCTTACTTGCT-3' were used. TransStartFastPfu DNA polymerase was used for polymerase chain reaction to obtain the CsrA desuccinylated gene amplified fragment and purified. (High-fidelity DNA polymerase produced by Beijing Quanshijin Biotechnology Co., Ltd. (TransGen Biotech)).

[0030] 2. Use The purified CsrA desuccinylation gene amplification fragment 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; purified CsrA desuccinylation gene amplification fragment 10-15ng; ExnaseMultis 2μL; ddH2O was added to the total system to 20μL. The CsrA desuccinylation recombinant vector was obtained by reaction and stored at -20℃ for future use.

[0031] 3. The recombinant vector obtained in step 2 was transformed into E. coli SM10 competent cells, and a single clone was selected for colony PCR verification. The successfully verified strain was transferred with Pseudomonas fluorescens at a ratio of 2:1-3.5:1. The successfully expressed strain was screened using kanamycin sulfate (Km), and a single clone was selected for PCR and sequencing verification to obtain a Pseudomonas fluorescens CsrA desuccinylated transformant (Pseudomonas fluorescens with high iron carrier production).

[0032] Example 3: Verification of high-yield iron carrier capability

[0033] 1. The CsrA desuccinylated transformant of Pseudomonas fluorescens obtained in step 2 and wild-type Pseudomonas fluorescens (i.e., Pseudomonas fluorescens PF08) were inoculated into MM9 culture medium at a volume ratio of 1:100, and cultured at 28°C and 200 rpm for 18 h. Total RNA was extracted with reagents, and genomic DNA was removed using DNase I. RT-PCR was used to detect the expression of siderophore synthesis and transport genes pvdA and pvdE, using this as a template. Figure 1 shown.

[0034] 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 h, measure the OD value at a wavelength of 630 nm, and calculate the siderophore production, such as Figure 2 shown.

[0035] The results are as follows Figure 1 and 2 As shown in the results, the expression levels of pvdA and pvdE and the siderophore production of the CsrA desuccinylated transformant of Pseudomonas fluorescens were significantly higher than those of wild-type Pseudomonas fluorescens (P < 0.05), and its siderophore production was as high as 89.4%, while the siderophore content of wild-type Pseudomonas fluorescens was 71.2%. Pseudomonas fluorescens regulates the synthesis of siderophores and the expression of transport proteins through the desuccinylation of CsrA protein, thereby promoting the production of siderophores.

[0036] In summary, the present invention clones the desuccinylation gene of the iron carrier regulatory protein CsrA into Pseudomonas fluorescens, and effectively promotes the synthesis and transport of Pseudomonas fluorescens iron carriers by highly expressing the desuccinylated CsrA protein, thereby achieving high production of iron carriers.

[0037] 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.

[0038] 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.

[0040]

[0041]

Claims

1. A method for constructing a fluorescent Pseudomonas strain with high iron carrier production, It is characterized in that The following steps are involved: S1: preparing a CsrA desuccinylation gene amplified fragment, the sequence of which is SEQ ID NO.1; S2: Construction of CsrA desuccinylation recombinant vector; S3: Construction of Pseudomonas fluorescens with high iron carrier production.

2. The construction method according to claim 1, It is characterized in that The steps of preparing the CsrA desuccinylation gene amplification fragment include extracting the whole genome DNA of Pseudomonas fluorescens as an amplification template; using CsrA (K38R) -F and CsrA (K38R) -R as amplification primers and using a high-fidelity enzyme for PCR amplification.

3. The construction method according to claim 2, It is characterized in that The sequence of primer CsrA(K38R)-F is SEQ ID NO.2, and the sequence of primer CsrA(K38R)-R is SEQ ID NO.

3.

4. The construction method according to claim 1, It is characterized in that The steps of constructing the CsrA desuccinylation recombinant vector include purifying the amplified fragment of the CsrA desuccinylation gene and cloning and recombining it into the vector pBBR1MCS-2 in one step.

5. The construction method according to claim 1, It is characterized in that The steps of constructing the fluorescent Pseudomonas with high iron carrier production include: transforming the CsrA desuccinylation recombinant vector into donor cells E.coli SM10, and screening to obtain positive donor cells; The positive donor cells are combined with the recipient cells Pseudomonas fluorescens to screen out Pseudomonas fluorescens with high iron carrier production.

6. The construction method according to claim 4, It is characterized in that The amount of CsrA desuccinylation gene amplified and purified fragment used is 10-15 ng, and the amount of vector pBBR1MCS-2 used is 100-110 ng.

7. The construction method according to claim 5, It is characterized in that The conjugation ratio of recipient cells to positive donor cells is 1:2-1:3.5, and the time of conjugation transfer is 16-36h.

8. The construction method according to claim 1, It is characterized in that The siderophore-producing organism is a hydroxamic acid-producing siderophore.

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