A method for rapidly screening high-yielding strains of antifungal active substance HSAF

Abstract

This invention discloses a method for rapidly screening high-yielding strains of the antifungal active substance HSAF. This method integrates low-dose extraction, high-throughput fermentation in microplates, specific assay culture, and plate confrontation detection. High-throughput fermentation of the strains is achieved using 48-well square plates, increasing throughput by tens of times compared to traditional shake flasks. *Pyrus pyrifolia* is selected as the specific assay bacterium, and a spore suspension with a concentration of 1×10³ CFU / mL is prepared. Based on the plate confrontation method, a clear inhibition zone forms within 12 hours, completing the rapid screening of high-yielding strains. Finally, HSAF is extracted using a low dose of 1 mL of fermentation broth, reducing reagent consumption by more than 60% compared to conventional methods. This invention is simple to operate and has good reproducibility. It is the first time, both domestically and internationally, that an integrated screening technology for high-yielding HSAF strains using ARTP mutagenesis has been established targeting *Bacillus OH11*, providing key technical support for the industrialization of HSAF.

Description

Technical Field

[0001] This invention belongs to the field of biological breeding and biological detection technology, specifically involving a rapid screening method for high-yield HSAF strains that integrates low-dose extraction, microplate fermentation, specific detection bacterial culture, and plate confrontation detection. It is suitable for the efficient screening of enzyme-producing OH11 mutant libraries and provides technical support for the industrialization of HSAF. Background Technology

[0002] Heat-stable antifungal factor (HSAF) is a natural antifungal active substance secreted by Lysobacter enzymogenes. It has a broad antibacterial spectrum (inhibiting fungi, oomycetes, nematodes, etc.), a unique mechanism of action (inhibiting the activity of pathogenic ceramide synthase), high thermal stability (>300℃), and low toxicity. Its chemical structure consists of a unique macrocyclic lactam system, a tetraamino acid structural unit, and a 5,5,6-tricyclic skeleton, which is different from existing commercial fungicides and is a core candidate component of novel green biological pesticides.

[0003]

[0004] HSAF chemical structure

[0005] Wild-type hygroscopic bacteria OH11 has an extremely low HSAF yield, approximately 1.6 mg / L. Although years of research have led to improvements in fermentation conditions and genetic engineering, resulting in laboratory-reported HSAF yields of 440-572 mg / L, this still falls far short of the g / L levels required for industrial production of commercially available agricultural antibiotics like avermectin. Traditional strain improvement methods have significant limitations: conventional physicochemical mutagenesis is indiscriminate and labor-intensive; while early genetic engineering techniques (such as introducing antibiotic resistance markers) may pose environmental and biosafety risks, and yield increases are prone to bottlenecks. Therefore, employing more precise and safer modern systems biotechnology strategies to overcome this yield challenge is crucial and urgent. New mutagenesis techniques such as ion beam implantation, space mutagenesis, and low-temperature plasma mutagenesis have emerged, with atmospheric and room-temperature plasma mutagenesis (ARTP) standing out due to its high safety, operational safety, and broad biocompatibility.

[0006] In the mutagenesis breeding technology system, the construction of the mutant library is only the initial step. The core issue for the efficient application of this technology is how to rapidly, accurately, and cost-effectively screen a small number of mutant strains with the desired superior phenotype from a massive pool of mutants. Specifically, for *Bacillus OH11*, there are three major challenges: First, the fermentation system has low throughput; traditional shake-flask fermentation can only process a small number of strains per batch, making it unsuitable for large-scale mutant libraries (typically containing thousands of mutant strains). Second, sample processing costs are high; conventional HSAF extraction requires more than 3 mL of fermentation broth, resulting in high reagent consumption and cumbersome operation. Third, the detection cycle is long; high-performance liquid chromatography (HPLC) quantitative detection of a single sample requires 30 minutes, making high-throughput screening difficult. These problems lead to long screening cycles, high costs, and low efficiency for high-yield HSAF strains, severely hindering the industrialization of HSAF.

[0007] Therefore, developing an integrated screening method based on low-dose extraction, high-throughput fermentation, and rapid detection is of great practical significance for shortening the breeding cycle and reducing screening costs. Summary of the Invention

[0008] The purpose of this invention is to address the shortcomings of existing screening technologies, such as low throughput, high cost, and slow detection, by providing a method for rapidly screening high-yield HSAF strains. This method achieves efficient and rapid screening of high-yield HSAF strains by optimizing the low-dose extraction process, microplate fermentation system, specific assay culture, and plate confrontation detection process.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A method for rapidly screening high-yielding strains of antifungal active substance HSAF includes the following steps:

[0011] (1) The control strain, enzyme-producing lysobacterium OH11, and the test strain were placed in different wells of a microplate and fermented to produce a fermentation broth containing HSAF.

[0012] (2) Spread the test fungus, Fusarium graminearum or anthracnose fungus, onto a plate;

[0013] (3) Drop the fermentation broth of each strain in step (1) onto the plate in step (2) and use the plate confrontation to preliminarily screen high-yield HSAF strains;

[0014] (4) Select candidate strains with an inhibition zone diameter greater than the control by more than 3 mm;

[0015] (5) Take 0.2-1 ml of culture medium from the candidate strain selected in step (4) in step (1), and use HPLC to quantitatively detect the HSAF content to select the high-yielding HSAF strain.

[0016] The fermentation broth mentioned in step (1) of this invention is the fermentation broth produced by the control strain and the test strain. The fermentation process can be carried out according to conventional methods in the art. The preferred specific fermentation process is as follows: the activated strain is inoculated into LB liquid medium and cultured at 28°C and 180 rpm for 12 h. The strain is then transferred into the fermentation medium of a microplate at an inoculation rate of 2.5% and cultured at 26~28°C and 170~180 rpm for 48~50 h.

[0017] The microplate mentioned in step (1) of this invention is a 24-well or 48-well plate, which can be round or square. Using microplate fermentation can significantly increase the number of fermenting strains, reduce reagent consumption and labor costs. The preferred microplate is a 48-well square plate.

[0018] The pathogens identified in step (2) of this invention are *Fusarium graminearum* and *Anthracnose pyrifolium*, preferably *Anthracnose pyrifolium*. Commercially available *Fusarium graminearum* and *Anthracnose pyrifolium* strains can be used for identification, such as *Fusarium graminearum* PH-1, *Fusarium graminearum* FG1, and *Anthracnose pyrifolium* NC40. More preferably, a spore suspension is spread onto a plate. The spore suspension can be obtained using conventional methods, preferably through fermentation on PDB medium, followed by centrifugation and washing with 0.85% NaCl. Using a spore suspension spreader instead of mycelium for plate antagonism can significantly shorten the inhibition zone formation time and accelerate the screening of high-yielding HSAF strains. The spore suspension concentration is 10... 3 CFU / mL.

[0019] The culture conditions for plate confrontation described in step (3) of the present invention are to culture in a constant temperature incubator at 25~28℃ for 12 to 96 hours, with the preferred culture time being 12 hours. At this time, a clear and stable inhibition zone can be formed on the plate.

[0020] The more HSAF produced by the test strain during fermentation, the larger the inhibition zone generated by plate confrontation. Therefore, step (4) selects candidate strains with a diameter greater than 3 mm than the control, which can achieve preliminary screening of high-yield strains, reduce the workload of subsequent HPLC detection, and achieve faster screening.

[0021] The inventors unexpectedly discovered that when the volume of the fermentation broth extracted was 0.2~1.0 mL, the detection results of the extracted HSAF were not significantly different from those of the standard 3 mL volume. Therefore, in step (5), 0.2~1.0 mL of culture medium can be selected for HPLC quantitative detection of the HSAF content. The preferred volume is 1 mL. Volumes lower than this range will increase the error caused by sample addition and solution evaporation.

[0022] The present invention (5) uses conventional methods in the field to quantitatively detect the content of HSAF by HPLC. In a specific example, the method is as follows: add an equal volume of ethyl acetate to the fermentation broth, add 5 μL of concentrated hydrochloric acid per milliliter of fermentation broth to adjust the pH to 2.0~5.0, vortex for 3 min, centrifuge at 10000 rpm for 3 min, take the supernatant and dry it in a fume hood, add 500 μL of methanol to dissolve it, filter it through a 0.22 μm organic filter, and then perform HPLC detection. The chromatographic column conditions and mobile phase elution conditions used for detection are shown in Table 1 and Table 2 below.

[0023] Table 1 Chromatographic conditions

[0024]

[0025] Table 2 Mobile phase elution conditions

[0026] .

[0027] The advantages of the method described in this invention compared to the prior art are as follows:

[0028] (1) High extraction efficiency and low cost: Small dose of fermentation broth (1 mL) extracts HSAF with an efficiency comparable to conventional 3 mL volume, reducing reagent consumption by more than 60% and lowering screening costs.

[0029] (2) The screening throughput is greatly improved: the 24 or 48-well plate fermentation system can culture dozens to hundreds of strains in parallel at one time, and the throughput is increased by dozens of times compared with the traditional shake flask, which is suitable for large-scale mutant library screening.

[0030] (3) Fast detection speed: Pear anthracnose spores are selected as the test bacteria, and the time for the inhibition zone to form is shortened to 12 hours, which is 80% shorter than the traditional mycelial block method (more than 4 days) and greatly improves the screening efficiency.

[0031] (4) High specificity and good repeatability: Pear anthracnose bacteria are highly sensitive to HSAF, have good spore uniformity, and clear inhibition zone boundaries, avoiding false positive or false negative results of traditional bacterial testing.

[0032] (5) Simple operation and wide applicability: The whole process does not require complicated instruments and can be carried out in a conventional laboratory. It is suitable for screening various enzyme-producing lysin mutant libraries. Attached Figure Description

[0033] Figure 1 HPLC analysis results of HSAF extracted from low-dose fermentation broth;

[0034] Figure 2 HPLC results of 24-well and 48-well plates with different liquid contents;

[0035] Figure 3 HPLC detection results of microplates with different plate types (24 wells and 48 wells);

[0036] Figure 4 HPLC detection results of microplates with different pore shapes (round holes, square holes);

[0037] Figure 5 HPLC results of 48-well plate and 250 mL Erlenmeyer flask;

[0038] Figure 6 Linear regression relationship between HSAF concentration and inhibition zone diameter;

[0039] Figure 7 Fermentation and microscopic examination results of various fungi;

[0040] Figure 8 Antagonistic results at different spore suspension concentrations (unit: CFU / mL).

[0041] Figure 9 Antagonistic results at different culture times;

[0042] Figure 10 ARTP-induced mutagenesis and secondary screening of HSAF yield. Detailed Implementation

[0043] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0044] The following examples use *Anthracnose fungus* (*Anthracnose cylindrica* NC40, published in Bao Yan, Biological Characteristics of *Anthracnose* and its Biocontrol and Antibacterial Activity, Master's Thesis, Nanjing Agricultural University), *Fusarium graminearum* (*Fusarium graminearum* PH-1, published in Gong Andong, Lei Yinyu, Li Xiang, Liu Jingrong, Li Xiangli, Hong Rui, Shen Hanbing, Zhang Yimei. Effects of FgFabD Gene on Growth, Development and Pathogenicity of *Fusarium graminearum* [J / OL]. Journal of Xinyang Normal University (Natural Science Edition). https: / / link.cnki.net / urlid / 41.1476.N.20251027.0856.002), *Alternaria alternata* (*Alternaria alternata* ACCC 37473), *Botrytis cinerea* (*Botrytis cinerea* ACCC 36028), and *Fusarium wilt* (*Fusarium oxysporum* cucumber-specific ACCC). 30220) and rice sheath blight fungus (Rhizoctonia solani 36124) are both known pathogenic strains, which are also preserved in our laboratory and are open to the public.

[0045] Unless otherwise specified, the components of each culture medium described in this invention are as follows:

[0046] ① Seed culture medium: LB medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L);

[0047] ② Fermentation medium: CDM502 medium ((NH4)2SO4 1 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, L-glutamate sodium 1.5 g / L, glucose 8 g / L, FeCl3 8.0 mg / L, pH 7.0-7.2).

[0048] ③ Culture media for testing bacteria: PDB liquid culture medium (potato 200 g / L, glucose 20 g / L), PDA solid culture medium (potato 200 g / L, glucose 20 g / L, agar 15 g / L).

[0049] The control strain L. enzymogenes OH11 used in the following examples was disclosed in 2007 (authorization announcement number CN101177671B). Preparation of the fermentation broth:

[0050] Seed culture preparation: Scrape a full loop of bacterial cells into 50 mL of LB medium and incubate at 28°C with shaking at 180 rpm for 12 h to obtain the seed culture.

[0051] Fermentation broth preparation: The OH11 seed culture was inoculated into a 250 mL shake flask (containing 50 mL CDM502 medium) and cultured at 28°C and 180 rpm for 48 h to obtain the standard fermentation broth. The standard fermentation broth used in the following examples was obtained by fermentation using the method of this embodiment.

[0052] Example 1: Feasibility verification of small-dose fermentation broth extraction of HSAF

[0053] Gradient volumetric extraction: 0.2 mL, 0.5 mL, 1 mL, and 3 mL of standard fermentation broth were taken respectively, and an equal volume of ethyl acetate was added. 5 μL of concentrated hydrochloric acid was added per mL of fermentation broth (to adjust the pH to 2.0–5.0). The mixture was vortexed for 3 min, centrifuged at 10,000 rpm for 3 min, and the supernatant was dried in a fume hood. The supernatant was dissolved in 500 μL of methanol, filtered through a 0.22 μm organic filter, and then analyzed by HPLC. The column conditions and mobile phase elution conditions used for the analysis are shown in Tables 1 and 2 below.

[0054] Table 1 Chromatographic conditions

[0055]

[0056] Table 2 Mobile phase elution conditions

[0057] .

[0058] Results: The detection results (characterized by chromatographic peak area) of the 0.2 ml, 0.5 ml, and 1.0 ml experimental groups were compared with the average value of the 3.0 ml control group, and bar charts were plotted. Figure 1 Visual analysis was performed. The data showed that there was no significant difference in the measured HSAF chromatographic peak area within the extraction volume range of 0.2 to 3.0 mL. The detection results of each trace volume group were highly consistent with the control group, proving that reducing the extraction volume to 0.2 mL to 1.0 mL did not affect the accuracy of HPLC quantitative analysis.

[0059] This result further demonstrates the feasibility of using micropore fermentation in this application.

[0060] Example 2: Microplate fermentation of HSAF

[0061] Liquid volume: In 24-well plates, liquid volume gradients of 0.6 mL, 1.0 mL, and 1.5 mL were set; in 48-well plates, liquid volume gradients of 0.6 mL, 0.8 mL, and 1.0 mL were set. After incubation at 28℃ and 180 rpm for 48 h, HSAF yield was measured.

[0062] Plate type: Use 24-well and 48-well plates with square and round holes, and the rest of the method is the same as the liquid filling volume.

[0063] Well type: Take 24-well and 48-well plates of the same type, with liquid volume of 0.6 mL and 1.0 mL respectively. The rest of the method is the same as the liquid volume optimization.

[0064] Feasibility verification: Take a 48-well square plate, fill it with 1 mL of liquid, and culture it under the same conditions as above.

[0065] Correlation between inhibition zone diameter and shake-flask potency: Test strains and control strain Bacillus OH11 with different HSAF yields were selected, and shake-flask fermentation was performed. HSAF was extracted according to the method used in Example 1, and the yield was detected by HPLC. The plate antagonism method of Example 4 was used for the experiment, and the diameter of the inhibition zone was measured. The HSAF yield of control strain Bacillus OH11 was used as the baseline (set as 100%), and the relative percentage of the yield of other strains was calculated and used as the abscissa. The measured diameter of the inhibition zone was used as the ordinate to draw a scatter plot and perform linear regression analysis.

[0066] Result: As Figure 2 , 34.5. When using microplate fermentation, changes in the liquid volume, plate shape, and well shape did not significantly affect the yield of HSAF. Comparing the performance of various microplate cultures of strain OH11 under different conditions, a 48-well square plate with a liquid volume of 1 mL was determined to be the optimal fermentation condition. Figure 6 It can be seen that there is a significant linear positive correlation between HSAF concentration (expressed as a relative percentage) and inhibition zone diameter. The coefficient of determination R² of the regression model is greater than 0.8, indicating that the difference in HSAF concentration can explain most of the variation in inhibition zone diameter, and the two are closely related. Therefore, the plate confrontation method is reliable for preliminary screening of high HSAF-producing strains.

[0067] Example 3: Selection of test bacteria and optimization of culture conditions

[0068] Screening of pathogens for detection: *Anthracnose fungus* (Cyclocarya paliurus NC40, published in Bao Yan, Biological Characteristics of *Anthracnose fungus* and its antibacterial activity, Master's thesis, Nanjing Agricultural University) was selected, along with *Fusarium graminearum* (PH-1, published in Gong Andong, Lei Yinyu, Li Xiang, Liu Jingrong, Li Xiangli, Hong Rui, Shen Hanbing, Zhang Yimei. Effects of FgFabD gene on growth, development and pathogenicity of *Fusarium graminearum* [J / OL]. Journal of Xinyang Normal University (Natural Science Edition). https: / / link.cnki.net / urlid / 41.1476.N.20251027.0856.002), *Alternaria alternata* (ACCC 37473), *Botrytis cinerea* (ACCC 36028), and *Fusarium oxysporum* (ACCC 37473), the causal agent of gray mold in tomatoes, and *Fusarium oxysporum* (ACCC 36028), the causal agent of wilt in cucumbers. Six fungi, including rice sheath blight pathogen (Rhizoctonia solani 36124), were inoculated into PDB medium and cultured at 28°C and 180 rpm for 72 h. The sporulation rate and sporulation uniformity were compared under a microscope.

[0069] Preparation of spore suspension: The pathogenic fungus on the prepared PDA plate was cut into small pieces and inoculated into PDB medium. The mixture was incubated at 28°C and 180 rpm for 72 h. Mycelia were removed by filtration through a sterile filter cloth. Spores were collected by centrifugation at 6000 rpm for 5 min and washed twice with 0.85% NaCl solution to obtain the spore suspension. The spore suspensions used in this embodiment were all obtained by fermentation using the method described in this embodiment.

[0070] Spore concentration: The spore suspension concentration was calculated using a hemocytometer, 10 5 10 4 10 3 10 2 CFU / mL serial dilution was performed, and the formation of inhibition zones was observed.

[0071] Incubation time: A suspension of pear anthracnose spores (1×10³ CFU / mL) was spread onto PDA plates and incubated at 28℃ for 12 h, 24 h, 48 h, 72 h, and 96 h, respectively. The formation and diameter stability of inhibition zones were observed. Results: From... Figure 7 It is known that only pear anthracnose fungus and wheat scab produce spores, especially pear anthracnose fungus, which has a stable spore production and good spore uniformity, while other fungi do not produce spores; Figure 8 It can be seen that excessively high spore concentrations inhibit the growth of the strain and prevent the formation of inhibition zones. As the spore concentration decreases, the inhibition zones gradually increase in size, reaching a maximum at 10... 3 The concentration of CFU / mL is the highest. Further reduction will prevent the bacterial colony from covering the plate and forming a clear and complete inhibition zone. Figure 9 It was observed that a clear inhibition zone formed after 12 hours of incubation, and the diameter of the inhibition zone stabilized after 24 hours. Thereafter, the inhibition zone showed no significant change with increasing incubation time. This indicates that the pear anthracnose pathogen selected in this invention is the optimal detection strain, and the optimal spore suspension concentration is 10. 3 The optimal detection time is 12 h of incubation at CFU / mL.

[0072] Example 4: Application of plate confrontation screening for high-yield HSAF strains

[0073] Mutant library preparation: Mutant strains were obtained by mutagenesis of enzyme-producing bacillus OH11 using ARTP at a power of 120 W, a helium flow rate of 10 SLM, an irradiation distance of 2 mm, and a treatment time of 50 s.

[0074] High-throughput screening: The control strain, Bacillus OH11, and the mutant strain were inoculated into 48-well square plates (1 mL CDM502 medium per well), fermented at 180 rpm for 48 h to obtain fermentation broth. 3.5 μL of fresh fermentation broth was spotted onto PDA plates coated with pear anthracnose spores (method as in Example 2), incubated at 28℃ for 12 h, and the diameter of the inhibition zone was measured to screen candidate strains with an inhibition zone diameter greater than 3 mm of the control strain.

[0075] Quantitative verification: 1 ml of culture medium was taken from the candidate strain and HPLC quantification was performed according to the method described in Example 1. The results are as follows. Figure 10 As shown, 21 high-yield strains were finally obtained, with yields increasing by 24.2%-169.8% compared to the original strain.

Claims

1. A method for rapidly screening high-yielding strains of antifungal active substance HSAF, characterized in that, Includes the following steps: (1) The control strain, enzyme-producing lysobacterium OH11, and the test strain were placed in different wells of a microplate and fermented to produce a fermentation broth containing HSAF. (2) Spread the test fungus, Fusarium graminearum or anthracnose fungus, onto a plate; (3) Drop the fermentation broth of each strain in step (1) onto the plate in step (2) and use the plate confrontation to preliminarily screen high-yield HSAF strains; (4) Select candidate strains with an inhibition zone diameter greater than the control by more than 3 mm; (5) Take 0.2-1 ml of culture medium from the candidate strain selected in step (4) in step (1), and use HPLC to quantitatively detect the HSAF content to select the high-yielding HSAF strain.

2. The method for rapidly screening high-yielding strains of antifungal active substance HSAF according to claim 1, characterized in that, The specific fermentation process of the fermentation broth in step (1) is as follows: the activated strain is inoculated into LB liquid medium and cultured at 28°C and 180 rpm for 12 h, and then transferred into the fermentation medium of microplate at an inoculation rate of 2.5% and cultured at 26~28°C and 170~180 rpm for 48~50 h.

3. The method for rapidly screening high-yielding strains of antifungal active substance HSAF according to claim 1, characterized in that, The micro-perforated plate mentioned in step (1) is a 24-hole or 48-hole plate, with round or square holes.

4. The method for rapidly screening high-yielding strains of the antifungal active substance HSAF according to claim 1, characterized in that, The bacteria identified in step (2) is *Pyracantha fortuneana*.

5. The method for rapidly screening high-yielding strains of antifungal active substance HSAF according to claim 1, characterized in that, The bacteria to be tested in step (2) are obtained by spreading a spore suspension onto a plate.

6. The method for rapidly screening high-yielding strains of antifungal active substance HSAF according to claim 5, characterized in that, The spore suspension was prepared by fermentation in PDB medium, followed by centrifugation and washing with 0.85% NaCl; preferably, the concentration of the spore suspension was 10. 3 CFU / mL.

7. The method for rapidly screening high-yielding strains of antifungal active substance HSAF according to claim 1, characterized in that, The culture conditions for the plate confrontation in step (3) are to culture in a constant temperature incubator at 25~28℃ for 12 to 96 hours, with the preferred culture time being 12 hours.

8. The method for rapidly screening high-yielding strains of antifungal active substance HSAF according to claim 1, characterized in that, Take 1 ml of culture medium from the candidate strain selected in step (4) in step (1), and use HPLC to quantitatively detect the HSAF content to select the high-yielding HSAF strain.

9. The method for rapidly screening high-yielding strains of antifungal active substance HSAF according to claim 1, characterized in that, The HPLC quantification method involved adding an equal volume of ethyl acetate to the fermentation broth, adding 5 μL of concentrated hydrochloric acid per milliliter of fermentation broth to adjust the pH to 2.0–5.0, vortexing for 3 min, centrifuging at 10,000 rpm for 3 min, taking the supernatant and drying it in a fume hood, dissolving it in 500 μL of methanol, filtering it through a 0.22 μm organic filter, and then performing HPLC detection.

10. The method for rapidly screening high-yielding strains of antifungal active substance HSAF according to claim 9, characterized in that, The chromatographic column conditions used for HPLC detection are as follows: ； The mobile phase elution conditions are as follows: 。

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