Strain for significantly improving yield of hsa f under iron limitation condition and application thereof

Abstract

The application discloses a strain for significantly improving HSAF yield under iron limitation and application thereof, and the genetically engineered bacterium is obtained by knocking out or silencing an iron response transcription factor Fur gene of L.enzymogenes OH11, and the nucleotide sequence of the Fur gene is shown as SEQ ID NO.1.Compared with a wild type strain, the mutant strain has no significant difference in growth in an iron deficiency or low-iron culture medium, and the HSAF synthesis capacity is greatly improved.The HSAF yield of the mutant strain in the iron deficiency culture medium reaches 162.34+5.84 mg / L, which is 3.60 times of that of the wild type strain.Therefore, the technical scheme of the application provides a more direct and effective way for improving the HSAF yield of L.enzymogenes OH11 in the iron deficiency or low-iron environment.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a strain that significantly increases HSAF production under iron-limited conditions and its applications. background

[0002] The genus *Lysobacterium* was established and classified by Christensen and Cook in 1978. Belonging to the phylum Proteobacteria, class Gammaproteobacteria, order Xanthomonadales, and family Xanthomonadaceae, it is a Gram-negative bacterium widely distributed in various natural environments. It exhibits excellent control effects against a variety of plant pathogenic fungi, bacteria, and nematodes, making it an important resource for the development of novel biological pesticides. To date, more than fifty species of *Lysobacterium* have been reported both domestically and internationally.

[0003] *Lysobacterium*, the type species of the genus *Lysobacterium*, obtains energy by degrading microorganisms and organic matter in the natural environment and is a saprophytic bacterium. *Lysobacterium* OH11, a novel biocontrol strain with independent intellectual property rights, was isolated from chili rhizosphere soil in our laboratory in 2006. It not only produces proteases, chitinases, β-1,3-glucanases, and cellulases, but also a variety of antibacterial small molecule compounds, especially heat-stable antifungal factor (HSAF), which plays a major role in its biocontrol mechanism. Compared with other antifungal substances, HSAF has two major advantages: first, its antibacterial mechanism is novel and non-toxic to mammals, insects, fish, and plants; second, it has high stability under heat, acid, and alkaline conditions, which is beneficial for industrialization and the extraction, transportation, storage, and use of HSAF. Over the past 20 years, our team has systematically conducted research on HSAF structural identification, synthetic pathways, genetic regulation, yield enhancement, and antibacterial mechanisms. However, for biocontrol bacteria, how to colonize in the natural environment and maximize their biocontrol activity is a major challenge in the current research and development of biocontrol products.

[0004] Iron is an essential nutrient for the survival of the vast majority of bacteria, participating in many important biological processes such as electron transport, respiration, DNA synthesis, and the tricarboxylic acid cycle. Although iron is the fourth most abundant element in the Earth's crust, most iron in nature exists in oxidized or hydroxide forms with extremely low solubility, and very little free iron exists. Therefore, enhancing the growth and metabolic capacity of biocontrol strains in iron-deficient or iron-limited environments is crucial for maximizing biocontrol activity. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a strain that significantly increases HSAF production under iron-limited conditions and its applications.

[0006] One objective of this invention is to provide a genetically engineered bacterium OH11-ΔFur, which is obtained by knocking out or silencing the Fur gene encoding the iron-responsive transcription factor from L. enzymogenes OH11, and the nucleotide sequence of the Fur gene is shown in SEQ ID NO.1.

[0007] The gene knockout or silencing method of the present invention can be carried out in accordance with conventional methods in the art. In some embodiments of the present invention, the knockout vector pEX18Gm-Fur is constructed and then the knockout strain is obtained by homologous recombination double exchange method. More specifically, for example, the electroporation and mutant screening method disclosed in our laboratory patent CN102943061A.

[0008] In one specific embodiment of the present invention, the following steps are included:

[0009] (1) Construction of the knockout vector

[0010] Primers for the upstream and downstream arms of the target gene Fur were designed. PCR amplification yielded approximately 600 bp fragments of homologous arms for each arm. Using a seamless cloning method, the Fur homologous arms were ligated to the suicide plasmid pEX18Gm. The resulting fragment was transformed into *Escherichia coli* DH5α. After verification by electrophoresis and sequencing, the fragment was named pEX18Gm-Fur and stored at -20℃ for later use. The suicide plasmid pEX18Gm was double-digested using Hind III / Xba I restriction enzymes.

[0011] (2) Obtaining mutant strains

[0012] The recombinant vector pEX18Gm-Fur was electroporated into wild-type lysozyme-producing Bacillus OH11 competent cells. The cells were plated on LB agar plates containing gentamicin for primary exchanger selection. Single colonies were picked and cultured in LB liquid for 7-9 hours for secondary exchange. 100 μL of the culture was plated on LB agar plates containing 10-15% sucrose. Secondary exchangers were picked and streaked simultaneously on gentamicin-resistant and non-resistant LB agar plates, and incubated at 28°C for 3-4 days. Exchangers that grew on non-resistant LB agar plates but not on gentamicin-resistant LB agar plates were verified by colony PCR, thus obtaining the gene knockout mutant, named OH11-ΔFur.

[0013] Unless otherwise specified, the molecular operations involved in the gene knockout methods described herein are all conventional methods in the field.

[0014] As an improvement, LB solid plates with a mass fraction of 10-15% sucrose in the secondary exchangers need to be supplemented with FeCl3 at a concentration of 10-50 mg / L, preferably 20-30 mg / L.

[0015] A second objective of this invention is to provide the application of the genetically engineered bacterium OH11-Δfur in the production of the antifungal active substance HSAF, particularly in the production of HSAF in iron-deficient or iron-limited environments.

[0016] In some embodiments, the specific method of the application described in this invention is to use the genetically engineered bacterium OH11-Δfur for fermentation culture. In some specific examples, the fermentation process is as follows: the mutant OH11-ΔFur is inoculated into LB liquid medium and cultured at 26-28°C and 170-190 rpm for 12-15 h. Then, it is transferred into fermentation medium at an inoculum of 1.0-2.5% and cultured at 26-28°C and 150-200 rpm for 48-60 h. The antifungal active substance HSAF in the fermentation broth is extracted and detected by HPLC.

[0017] In some embodiments, the fermentation medium of the present invention comprises: glucose 5-15 g / L, (NH4)2SO4 0.5-2.0 g / L, FeCl3 0-32 mg / L, MgCl2 5-20 mg / L, K2HPO4 0.5-4.0 g / L, KH2PO4 0.25-2.0 g / L, and CaCO3 0.25-1.5 g / L. Preferably, the fermentation medium comprises: glucose 5-10 g / L, (NH4)2SO4 0.8-1.2 g / L, FeCl3 0-8 mg / L, MgCl2 8-15 mg / L, K2HPO4 1.0-1.5 g / L, KH2PO4 0.5-0.75 g / L, and CaCO3 0.5-1.0 g / L. A more preferred fermentation medium composition is: glucose 8 g / L, (NH4)2SO4 1.0 g / L, MgCl2 10 mg / L, K2HPO4 1.0 g / L, KH2PO4 0.5 g / L, and CaCO3 1.0 g / L. All fermentation media are prepared with deionized water.

[0018] In some embodiments, the extraction of the antifungal active substance HSAF from the fermentation broth by the present invention specifically involves taking the fermentation broth, adding concentrated HCl dropwise until the pH is 2.8-3.0, adding CaCl2 and ethyl acetate, vortexing to ensure that the organic solvent and the fermentation broth are in full contact, centrifuging to separate the fermentation broth and the organic solvent into two phases, and the supernatant is the HSAF organic solvent layer.

[0019] In some embodiments, the amount of CaCl2 added is 0.1–0.2 g / mL of fermentation broth.

[0020] In some embodiments, the volume ratio of ethyl acetate to fermentation broth is 1:1.

[0021] The vortex oscillation described in this invention can be a conventional method in the art, such as placing it on a vortex mixing oscillator and oscillating at 2000 rpm for 1 minute.

[0022] The centrifugation of this invention can be performed according to conventional methods in the art, such as centrifugation at 8000 rpm for 5 min.

[0023] The present invention can be detected by HPLC using the HSAF organic solvent layer. For example, 1 mL of the HSAF organic solvent layer can be dried, dissolved in 500 μL of methanol, and then the HSAF content can be detected by HPLC.

[0024] HPLC detection conditions: InterSustainSwift C18 5μm, 250×4.6mm. Mobile phase: Solution A (0.025% TFA aqueous solution) and Solution B (0.025% TFA acetonitrile solution), flow rate: 1mL / min; injection volume: 20μL; UV absorbance: 318nm; injection program: 0-10min, increasing solution B from 5% to 25%; 25min, increasing to 80%; 26min, increasing to 100%; 30min, returning to 5%, with the total ratio of solutions A and B being 100%; peak area was recorded. The linear relationship between HSAF concentration and peak area was also established as Y = 4E - 0.5X + 12.46, R0 2 The content of HSAF in ethyl acetate is calculated using a value of 0.9996.

[0025] A third objective of this invention is to provide a gene Fur that encodes an iron-responsive transcription factor, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0026] The present invention also provides the application of the gene Fur in increasing the production of the antifungal active substance HSAF by L. enzymogenes OH11 in iron-deficient or iron-limited environments; the application specifically involves knocking out or silencing the gene Fur in L. enzymogenes OH11.

[0027] The advantages of this invention over the prior art are:

[0028] (1) The present invention successfully constructed an HSAF-producing strain OH11-ΔFur through molecular biology technology. Compared with the wild-type strain, the genetically engineered strain OH11-ΔFur of the present invention showed no significant difference in growth in iron-deficient or low-iron culture media, while the HSAF synthesis capacity was greatly improved. In some examples, the HSAF yield reached 162.34±5.84 mg / L, which is 3.60 times that of the wild-type strain.

[0029] (2) The genetically engineered bacterium OH11-ΔFur of the present invention significantly increases the yield of HSAF under iron-limited conditions, and can give full play to the antagonistic activity of the strain in iron-deficient environment. It provides a more direct and effective way to increase the yield of HSAF' of enzyme-producing bacterium OH11 in iron-deficient or low-iron environment. It is of great significance for the practical application of biocontrol agents in the field and lays the foundation for future field application. Attached Figure Description

[0030] Figure 1 Construct the vector bacterial culture for PCR validation (lanes 1-12 are E. coli positive transformants, M is the standard);

[0031] Figure 2 PCR verification of mutant strains; (lanes 1-2 are OH11 mutant strains, CK is OH11 wild-type strain, and M is standard).

[0032] Figure 3 Fermentation of mutant strain OH11-ΔFur in iron-deficient synthetic medium;

[0033] Figure 4 Fermentation of mutant strain OH11-ΔFur in iron-containing synthetic medium;

[0034] Figure 5 Fermentation of mutant strain OH11-ΔFur in 10% TSB medium. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of protection of the present invention.

[0036] Unless otherwise specified, the molecular experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] Example 1: Construction of the gene Fur knockout vector pEX18-Fur

[0039] First, based on the genome information of *Bacillus OH11*, the upstream and downstream homologous arm sequences of the gene *Fur* were retrieved (as shown in SEQ ID NO: 2 and 3 of the sequence listing), each approximately 600 bp. Simultaneously, the multiple cloning site sequence of the suicide plasmid pEX18Gm was retrieved (as shown in SEQ ID NO: 4 of the sequence listing). Using a seamless cloning primer design tool (https: / / soft.transgen.com.cn / ), the upstream and downstream arm primers for *Fur* were designed online, as shown below:

[0040] Primers used to amplify the upstream homologous arm of Fur:

[0041] Fur-up-fw:5'-cgacggccagtgccaagcttACTTGGTCAGCGCGTCGTT-3′

[0042] Fur-up-rev:5'-ggtcgcgacgcggccGGGGGTACGTCCCTTCGA-3'

[0043] Primers used to amplify the downstream homologous arm of Fur:

[0044] Fur-dw-fw:5'-aagggacgtacccccGGCCGCGTCGCGAC-3'

[0045] Fur-dw-rev:5'-gtacccggggatcctctagaCGTCCGGGGGGAGCAT-3'.

[0046] Using the genome of *Bacillus OH11*, an enzyme-producing bacterium, as a template, PCR amplification was performed using TransGen Biotech's TransStartFast DNA Polymerase. The fragments, Fur-up and Fur-dw, representing the upstream and downstream homologous arms of the *Fur* gene, were detected and recovered by agarose gel electrophoresis. Simultaneously, the suicide plasmid pEX18Gm was double-digested with Hind III / Xba I to obtain a linearized plasmid fragment. This linearized fragment, along with the Fur-up and Fur-dw fragments, was cloned using the pEASY-Basic Seamless Cloning and Assembly Kit. The clones were then transformed into *Escherichia coli* DH5α, and transformants were selected for agarose gel electrophoresis verification. Figure 1 As shown, an electrophoretic band appeared at 1200 bp, preliminarily indicating that the upper and lower homologous arms Fur-up and Fur-dw were successfully ligated to plasmid pEX18Gm. Further, after sequencing verification, it was named pEX18Gm-Fur and stored at -20℃ for later use.

[0047] Example 2: Obtaining the mutant strain OH11-ΔFur

[0048] The recombinant vector pEX18-Fur was transformed into Bacillus OH11 competent cells using an electroporation method. For specific methods and preparation of competent cells, please refer to patent CN102943061A. 100 μL of cells were spread onto LB agar plates containing 100 μg / mL gentamicin resistance and incubated at 28°C for 4 days. A single colony of the primary exchanger was picked and placed in 1 mL of LB liquid and cultured at 28°C with shaking at 180 rpm for 7 hours. For the secondary exchanger, 100 μL of the secondary exchanger was spread onto LB agar plates containing 15% sucrose and 30 mg / L FeCl3 and incubated at 28°C for 3 days. The secondary exchanger was then picked and streaked onto both LB agar plates containing 100 μg / mL gentamicin resistance and non-resistant LB agar plates and incubated at 28°C for 2 days. Only colonies that grew on non-resistant LB agar plates but not on gentamicin-resistant LB agar plates were considered potential secondary exchangers. Colonies were verified by PCR using primers Fur-up-fw and Fur-dw-rev. If the PCR verification result of the transformant shows a band at 1600 bp, it indicates a gene reversion mutation, and the transformant is *Bacillus OH11*, which produces enzymes; if the electrophoresis band shows a band at 1200 bp, it indicates that the *Fur* gene has been successfully knocked out, and the transformant is a positive transformant (see...). Figure 2 ); Subsequently, further sequencing verification was performed on the positive transformants, and the Fur mutant strain with successful double crossover was obtained and named OH11-ΔFur.

[0049] Example 3: Fermentation culture of mutant strain OH11-ΔFur in iron-deficient synthetic medium

[0050] The mutant strain OH11-ΔFur was inoculated into 100 mL of LB liquid medium and cultured at 28 °C with shaking at 180 rpm for 12 h to obtain the seed culture. The seed culture was then transferred to 100 mL of fermentation medium at an inoculation rate of 2.5% and cultured at 28 °C with shaking at 180 rpm for 48 h to obtain the HSAF fermentation broth. Finally, HSAF was extracted from the fermentation broth and detected by HPLC. The wild-type strain OH11 was used as a control.

[0051] The fermentation medium consists of: 8 g / L glucose, 1.0 g / L (NH4)2SO4, 10 mg / L MgCl2, 1.0 g / L K2HPO4, 0.5 g / L KH2PO4, and 1.0 g / L CaCO3. The medium is prepared with deionized water.

[0052] The HSAF extraction and detection steps were as follows: 3 mL of fermentation broth was transferred to a 15 mL centrifuge tube, concentrated HCl was added dropwise to bring the pH to 3.0, 0.45 g CaCl2 and 3 mL ethyl acetate were added to the mixture, and the mixture was vortexed at 2000 rpm for 1 min to ensure sufficient contact between the organic solvent and the fermentation broth. The mixture was then centrifuged at 8000 rpm for 5 min to separate the fermentation broth and organic solvent into two phases. The supernatant was the HSAF organic solvent layer. 1 mL of this layer was dried, dissolved in 500 μL of methanol, and its content was detected by HPLC. HPLC detection conditions: InterSustainSwift C18 5 μm, 250 × 4.6 mm. Mobile phase: Solution A (0.025% TFA aqueous solution) and Solution B (0.025% TFA acetonitrile solution); flow rate: 1 mL / min; injection volume: 20 μL; UV absorbance: 318 nm; injection program: 0-10 min, increasing solution B from 5% to 25%; 25 min, increasing to 80%; 26 min, increasing to 100%; 30 min, returning to 5%, with the total ratio of solutions A and B being 100%; peak area recorded; the linear equation between HSAF concentration and peak area was established as Y = 4E - 0.5X + 12.46, R... 2 The content of HSAF in ethyl acetate is calculated using a value of 0.9996.

[0053] The results are as follows Figure 3 As shown, compared with the wild-type strain OH11, the deletion of the Fur gene did not significantly affect the growth of strain OH11-ΔFur, while the HSAF yield increased from 45.06±5.94 mg / L to 162.34±5.87 mg / L, an increase of 3.60 times. Therefore, the technical solution of this invention can effectively improve the ability of enzyme-producing Bacillus OH11 to synthesize HSAF in an iron-deficient environment, which is of great significance for the subsequent field application of biocontrol agents.

[0054] Example 4: Fermentation culture of mutant strain OH11-ΔFur in iron-containing synthetic medium

[0055] Fermentation media containing different concentrations of FeCl3 (2 mg / L, 4 mg / L, 8 mg / L, 16 mg / L, and 32 mg / L), with other components as described in Example 3, were prepared and dispensed into 500 mL Erlenmeyer flasks (100 mL per flask). The flasks were sterilized at 121°C for 15 min and then cooled for later use. Wild-type and mutant strains were simultaneously cultured for fermentation. The specific fermentation process and the detection of HSAF yield in the fermentation broth were as described in Example 3.

[0056] The results are as follows Figure 4As shown, the growth of the mutant strain OH11-ΔFur was not significantly different from that of the wild-type strain OH11 after adding different concentrations of FeCl3. In fermentation medium with low concentrations of FeCl3 (≤8 mg / L), the deletion of the Fur gene promoted HSAF production to some extent; when the FeCl3 concentration exceeded 8 mg / L, the HSAF production of OH11-ΔFur was not significantly different from that of the wild type. This indicates that the promotion of HSAF synthesis by the Fur gene is regulated by iron ions in the culture medium.

[0057] Example 5: Fermentation culture of mutant strain OH11-ΔFur in 10% TSB medium

[0058] Prepare 10% TSB natural culture medium, dispense 100 mL into 500 mL Erlenmeyer flasks, sterilize at 121°C for 15 min, and cool for later use. Simultaneously ferment the wild-type strain and the mutant strain. The specific fermentation process and the detection of HSAF yield in the fermentation broth are as described in Example 3.

[0059] The results are as follows Figure 5 As shown: In natural culture medium, the growth and HSAF synthesis capacity of mutant strain OH11-ΔFur were not significantly different from those of wild-type OH11. This is because the natural culture medium contains a certain concentration of iron ions, and the promotion of HSAF synthesis by Fur is regulated by the iron ions in the culture medium, as described in Example 4.

Claims

1. A genetically engineered bacterium OH11-Δ Fur Its characteristics are, The genetically engineered bacteria is an enzyme-producing lysobacterium (Bacillus lysinus). L. enzymogenes OH11 knockout or silencing encodes iron-responsive transcription factors Fur The gene was obtained after that, the Fur The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The genetically engineered bacterium OH11-Δ according to claim 1 Fur Its characteristics are, Gene knockout was performed using the constructed knockout vector pEX18Gm- Fur Then, the knockout strain was obtained using the homologous recombination double exchange method.

3. The genetically engineered bacterium OH11-Δ according to claim 1 or 2 fur Application in the production of the antifungal active substance HSAF.

4. The genetically engineered bacterium OH11-Δ as described in claim 1 or 2 fur Application in the production of antifungal active substance HSAF in iron-deficient or iron-limited environments.

5. The application according to claim 3 or 4, characterized in that, It utilizes the genetically engineered bacteria OH11-Δ fur Fermentation culture is carried out.

6. The application according to claim 5, characterized in that, Fermentation culture involves using the mutant OH11-Δ Fur Inoculate into LB liquid medium and culture at 26–28°C and 170–190 rpm for 12–15 h. Then, transfer to fermentation medium at an inoculum rate of 1.0–2.5% and culture at 26–28°C and 150–200 rpm for 48–60 h. Extract the antifungal active substance HSAF from the fermentation broth.

7. The application according to claim 6, characterized in that, The fermentation medium consists of: glucose 5-15 g / L, (NH4)2SO4 0.5-2.0 g / L, FeCl3 0-32 mg / L, MgCl2 5-20 mg / L, K2HPO4 0.5-4.0 g / L, KH2PO4 0.25-2.0 g / L, and CaCO3 0.25-1.5 g / L.

8. The application according to claim 7, characterized in that, The fermentation medium consists of: glucose 5-10 g / L, (NH4)2SO4 0.8-1.2 g / L, FeCl3 0-8 mg / L, MgCl2 8-15 mg / L, K2HPO4 1.0-1.5 g / L, KH2PO4 0.5-0.75 g / L, and CaCO3 0.5-1.0 g / L.

9. The application according to claim 8, characterized in that, The fermentation medium consists of: 8 g / L glucose, 1.0 g / L (NH4)2SO4, 10 mg / L MgCl2, 1.0 g / L K2HPO4, 0.5 g / L KH2PO4, and 1.0 g / L CaCO3.

10. The application according to claim 6, characterized in that, The extraction of antifungal active substance HSAF from fermentation broth involves taking the fermentation broth, adding concentrated HCl dropwise until the pH is 2.8–3.0, adding CaCl2 and ethyl acetate, vortexing to ensure full contact between the organic solvent and the fermentation broth, centrifuging to separate the fermentation broth and organic solvent into two phases, with the supernatant being the HSAF organic solvent layer.

11. The application according to claim 10, characterized in that, The amount of CaCl2 added is 0.1-0.2 g / mL of fermentation broth; the volume ratio of ethyl acetate to fermentation broth is 1:

1.

12. Genes as shown in SEQ ID NO.1 Fur In iron-deficient or iron-limited environments, enzyme-producing lysinophils ( L. enzymogenes Applications of OH11 in producing the antifungal active substance HSAF; specifically, the application involves knocking out or silencing enzyme-producing bacilli ( L. enzymogenes Genes in OH11 Fur .

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