Genetically engineered bacterium for resisting bacillus velezensis as well as preparation method and application of genetically engineered bacterium

Abstract

The invention belongs to the technical field of genetically engineered bacteria, and particularly discloses an anti-penicillium velezensis genetically engineered bacterium as well as a preparation method and application thereof. According to the genetically engineered bacterium, a polyketide synthase gene cluster in a genome of bacillus velezensis is knocked out or inactivated, and the gene cluster comprises one of PKS5, PKS6 and PKS10 gene clusters. Through specific knockout of a polyketide synthase gene cluster competing with a target product, the metabolic flux is redistributed while the excellent growth performance of the strain is reserved, the yield of the anti-penicillium active substance is remarkably improved, and a new technical scheme is provided for solving the bottleneck of low strain titer in the existing biological control technology. Compared with an original strain, the bacillus subtilis has the advantages that the utilization efficiency of nutrient substances and the adaptability to the fermentation environment in industrial production are enhanced, the inhibition effect on penicillium is improved by 10.4%, and the bacillus subtilis has a good application prospect in the aspect of increasing the yield of industrial anti-penicillium active substances.

Description

Technical Field

[0001] This invention relates to the field of genetically engineered bacteria, and in particular to a genetically engineered strain of Bacillus bellis resistant to Penicillium, its preparation method, and its application. Background Technology

[0002] Mold growth is a core cause of spoilage in fruits, vegetables, grains, and various foods, directly impacting people's daily lives and food safety. Molds possess an extremely strong reproductive capacity, primarily reproducing through diverse asexual or sexual spores. The sheer number of spores is a key factor in their rapid spread and large-scale proliferation. More importantly, some molds produce carcinogenic or other toxic mycotoxins during their growth, which, once ingested through food, pose a serious threat to human health. In recent years, fungal contamination of agricultural products and food, especially mold and mycotoxin contamination, has received widespread global attention, making mold control a core research topic in food and feed hygiene.

[0003] According to Li Can's review in "Research Progress on Degradation Methods of Mycotoxins in Feed", the current methods for controlling molds and toxins mainly include physical methods, chemical methods and biological methods.

[0004] Limitations of existing technology: 1. Although physical methods (such as adsorbents and heat treatment) are widely used, they are often accompanied by non-specific loss of feed nutrients (such as vitamins and minerals), and the adsorption efficiency often fluctuates with the type of toxin.

[0005] 2. Although chemical methods (such as ammoniation and oxidation) are fast-acting, they can easily damage the palatability of feed, and residual chemical reagents may pose food safety risks and cause secondary pollution.

[0006] 3. Biological methods (utilizing microorganisms or enzymes) are considered the most promising approach due to their high specificity, safety, and lack of residue. However, most currently used biological agents are wild-type strains (such as wild-type Bacillus and yeast), whose genomes contain a large number of non-essential secondary metabolic gene clusters. These redundant gene clusters not only fail to produce the target antibacterial substance but also competitively consume substrates and cellular energy, resulting in low synthesis efficiency and unstable yield of the target antifungal active substance, making it difficult to meet the industrial application requirements for low cost and high efficiency. Summary of the Invention

[0007] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing a genetically engineered strain of *Bacillus belyssima* resistant to *Penicillium*, its preparation method, and its application. By knocking out redundant polyketide synthase (PKS) gene clusters in the genome, redistributing cell resources, and combining with an optimized fermentation process, the strain's ability to synthesize active substances against *Penicillium* is significantly improved.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention is to provide a genetically engineered strain of Bacillus berleis, wherein a cluster of polyketide synthase genes in the genome of Bacillus berleis is knocked out or inactivated, the cluster of genes including one of the PKS5, PKS6, and PKS10 gene clusters.

[0009] Furthermore, the gene cluster that is knocked out or inactivated is PKS6.

[0010] Furthermore, the Bacillus belyssus has the accession number CGMCC No. 36999.

[0011] Furthermore, the Bacillus velezensis was deposited on December 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as Bacillus velezensis with accession number CGMCC No:36999. Furthermore, based on the whole genome sequence positions of the *Bacillus belyssus*, the pks5 gene cluster is located at positions 1426080-1514310 of the whole genome sequence; the pks6 gene cluster is located at positions 1735334-1845433 of the whole genome sequence; and the pks10 gene is located at positions 2475831-2496721 of the whole genome sequence.

[0012] A second aspect of the present invention is to provide a method for constructing a genetically engineered bacterium resistant to Penicillium berberis, comprising the following steps: Provide the above-mentioned Bacillus belysinus; Gene manipulation elements were designed to knock out or inactivate a polyketide synthase gene cluster in the genome of the Bacillus belyssus. The gene manipulation element is introduced into the Bacillus belyssus, causing the polyketide synthase gene cluster to be knocked out or inactivated. Obtain Bacillus belyss genetically engineered bacteria in which the polyketide synthase gene cluster has been knocked out or inactivated.

[0013] Furthermore, the gene manipulation elements include homologous recombination fragments, CRISPR / Cas gene editing systems, transposon elements, or combinations thereof.

[0014] Furthermore, the polyketide synthase gene cluster is one of the PKS5, PKS6, and PKS10 gene clusters.

[0015] A third aspect of the present invention is to provide a fermentation method for an anti-Penicillium berberis genetically engineered bacterium, wherein the bacterium is inoculated into a culture medium for fermentation culture to obtain a target fermentation product. The culture medium has the following formulation: corn starch 8-12 g / L, yeast extract 20-30 g / L, manganese sulfate 0.001-0.01 g / L, potassium dihydrogen phosphate 0.3-0.7 g / L, pH 7.0-8.0.

[0016] Furthermore, the amount of inoculation is 2% to 5% of the culture medium, and fermentation is carried out at 30°C to 37°C for 48 to 72 hours.

[0017] A fourth aspect of the present invention is to provide the use of the fermentation product obtained by the above-described fermentation method of Bacillus belye genetically engineered bacteria in the preparation of a microbial preparation for inhibiting the growth of Penicillium.

[0018] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention, through genome simplification of Bacillus belyssus and specific knockout of the polyketide synthase (PKS) gene cluster competing with the target product, significantly increases the yield of anti-Penicillium active substances by redistributing metabolic flux while preserving the excellent growth performance of the strain. This provides a new technical solution to address the bottleneck of "low strain potency" in existing biocontrol technologies. Compared to the starting strain, it enhances the efficiency of nutrient utilization and adaptability to the fermentation environment in industrial production, while still maintaining good growth and fermentation performance; it has good application prospects in increasing the yield of anti-Penicillium active substances in industrial production. (2) This invention improves the yield of anti-Penicillium active substances synthesized by Bacillus belyssus by optimizing the fermentation medium (carbon source, nitrogen source, temperature) to varying degrees. The inhibition zone method of fermentation broth was used to compare the inhibitory effects of the modified strain and the original strain on Penicillium. The average diameter of the recombinant Bacillus belyssus genetically engineered strain can reach 24.3 mm, and the inhibition rate is increased by 10.4% compared with the original strain.

[0019] (3) Gene knockout did not affect the normal growth performance of the strain, and the engineered strain retained good fermentation adaptability.

[0020] (4) The optimized fermentation medium (corn starch + yeast extract system) and fermentation conditions (37℃, pH 7.5) further improved the yield and have the potential for industrial application.

[0021] (5) This strain is derived from commonly used plant rhizosphere growth-promoting bacteria, and its fermentation products can be used as green biological preservatives to solve the problem of fungal contamination of agricultural products. Attached Figure Description

[0022] Figure 1Microscopic images and single colony morphology of Bacillus belyssus TK1926; Figure 2 Analysis diagram of gene clusters encoding antimicrobial secondary metabolites of Bacillus belyssus TK1926 and location diagram of PKS5, PKS6, and PKS10 gene clusters; Figure 3 The plasmid map and construction process diagram of the temperature-sensitive knockout vector PKSVT; Figure 4 To verify the PKS5, PKS6, and PKS10 gene cluster knockout, plasmid linearization, and homologous recombination gel electrophoresis results by PCR; Figure 5 Comparison of growth curves of Bacillus belyssus TK1926 and various knockout strains in LB medium; Figure 6 The inhibition zone diagram and bar chart show the antibacterial activity of the fermentation broth of the engineered strain under fermentation medium A. Figure 7 The inhibition zone diagram and bar chart show the antibacterial activity of the fermentation broth of the engineered strain under fermentation medium B. Figure 8 The inhibition zone diagram and bar chart are shown for the antibacterial activity of the fermentation broth of the engineered strain under fermentation medium C. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0024] This invention provides a genetically engineered strain of Bacillus belyceae, in which a polyketide synthase (PKS) gene cluster has been knocked out in the genome, preferably one of the PKS5, PKS6, or PKS10 gene clusters. This engineered strain retains good growth performance while significantly enhancing its ability to synthesize Penicillium-resistant active substances.

[0025] Preferably, the original strain of Bacillus velezensis is Bacillus velezensis TK1926 (accession number: CGMCC No. 36999), such as Figure 1 When the host bacterium is Bacillus velezensis TK1926: The pks5 gene cluster is located at positions 1426080-1514310 of the host bacterium's whole genome sequence. The pks6 gene cluster is located at positions 1735334-1845433 on the host bacterium's whole genome sequence. The pks10 gene is located at positions 2475831-2496721 of the host bacterium's whole genome sequence, corresponding to the genome sequence position encoding *Bacillus belyssus* TK1926 (accession number: CGMCC No. 36999) in GenBank. Its encoded product participates in the biosynthesis of polyketide secondary metabolites, such as... Figure 2 .

[0026] The present invention also provides a method for constructing the above-mentioned Bacillus belyss genetically engineered strain, comprising: firstly constructing gene knockout plasmids PKSVT-Δpks5, PKSVT-Δpks6, and PKSVT-Δpks10; electroporating the knockout plasmids into Bacillus belyss; and screening to obtain recombinant Bacillus belyss Δpks5, Δpks6, and Δpks10 with the polyketide synthase gene knocked out.

[0027] Preferably, the gene knockout plasmids PKSVT-Δpks5, PKSVT-Δpks6, and PKSVT-Δpks10 are... The construction methods include: 1-1) Amplify homologous sequences of the target gene Using the genome of Bacillus belye TK1926 as a template, PCR was performed to amplify the upstream homologous arm fragments pks5-up, pks6-up, and pks10-up and the downstream homologous arm fragments pks5-down, pks6-down, and pks10-down of the key region of the polyketide synthase gene, respectively. The sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0028] PCR amplification was performed using Bacillus belye genome as a template; the amplification reaction system is shown in Table 1 below: Table 1. Amplification reaction system.

[0029]

[0030] The amplification program was set as follows: pre-denaturation: 98℃ for 2 min; denaturation: 98℃ for 10 s; annealing: 60℃ for 5 s; extension: 72℃ for 25 s; 35 cycles; extension: 72℃ for 5 min.

[0031] The PCR products were subjected to agarose gel electrophoresis. The size of the up and down bands was about 500 bp. The PCR products, i.e., the upstream and downstream homologous arm fragments of the target gene, were then recovered using a DNA gel recovery kit.

[0032] 1-2) Linearization of expression vectors The PKSVT plasmid (sequence shown in SEQ ID NO:1) was extracted according to the kit's instruction manual. After double digestion with SmaI and SacI, the product was subjected to agarose gel electrophoresis, and then recovered using a DNA gel recovery kit to obtain the linearized vector sequence.

[0033] The double enzyme digestion system is shown in Table 2 below: Table 2. Double enzyme digestion system.

[0034]

[0035] After mixing, the enzyme was digested in a 37°C water bath for 30 min. After the reaction was completed, the enzyme digestion product was subjected to agarose gel electrophoresis. The band was about 6000 bp. The enzyme digestion product was then recovered using a DNA gel recovery kit: linear PKSVT plasmid.

[0036] 1-3) The upstream and downstream homologous arm fragments of the polyketide synthase gene obtained in step 1-1) are ligated using overlap extension PCR to obtain the insert fragment; 1-4) Perform a one-step seamless cloning ligation reaction on the insert obtained in steps 1-3) and the plasmid PKSVT linearized with restriction endonucleases SamⅠ and SacⅠ, as shown in Table 3; after mixing evenly, react in a water bath at 50℃ for 15 min.

[0037] Table 3. Connection reaction system.

[0038]

[0039] 1-5) The ligation products obtained in steps 1-4) were transformed into competent JM109 E. coli cells, screened on LB plates with 50 μg / ml kanamycin resistance, and confirmed by PCR and sequencing. Knockout plasmids PKSVT-Δpks5, PKSVT-Δpks6, and PKSVT-Δpks10 were obtained.

[0040] This invention also provides a Bacillus belyceae genetically engineered strain with increased yield of the aforementioned anti-Penicillium active substances. Applications in microbial fermentation.

[0041] Preferably, the application includes inoculating a seed culture prepared from a genetically engineered strain of Bacillus belye into a fermentation medium to produce antifungal active substances.

[0042] The fermentation medium consists of a carbon source, a nitrogen source, and inorganic salts, wherein the carbon source includes any one of corn starch, sucrose, or glucose; Nitrogen sources include any one of beef extract or yeast extract; Inorganic salts include any one or a combination of several of the following: dipotassium hydrogen phosphate, manganese sulfate, magnesium sulfate, and calcium carbonate. The fermentation conditions in the fermentation medium were: temperature 30 / 37℃, pH 6-8, and rotation speed 220 rpm.

[0043] More preferably, the basic fermentation medium formula is: 10 g / L glucose, 10 g / L yeast extract, 0.5 g / L potassium dihydrogen phosphate, 0.005 g / L manganese sulfate, 0.05 g / ml magnesium sulfate, and 0.1 g / ml calcium carbonate.

[0044] The basic fermentation conditions are: pH=7.6 and temperature 30℃.

[0045] More preferably, the recombinant Bacillus belye genetically engineered strain is activated on an LB plate until single clones are grown. These single clones are then picked and cultured in LB liquid test tubes, and then transferred to seed culture medium and cultured until the optical density OD600 value reaches between 1.5 and 2 to obtain a seed solution. The seed solution is then inoculated into fermentation medium at a rate of 2% (volume percentage) to obtain a fermentation broth containing fermentation products. The seed culture medium has the following formulation: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with deionized water as the solvent. The culture conditions in the seed culture medium were: 37℃, 220rpm for 8-12 h.

[0046] Those skilled in the art can implement the technical solutions of the present invention based on the methods described herein.

[0047] The invention has now been generally described, and will be more readily understood by referring to the following embodiments, which are provided by way of example and not by way of limitation.

[0048] The original strains of Bacillus belyssus were all Bacillus belyssus TK1926; the PKSVT plasmid was obtained from the laboratory. The basic fermentation medium formula is: 10 g / L glucose, 10 g / L yeast extract, 0.5 g / L potassium dihydrogen phosphate, 0.005 g / L manganese sulfate, 0.05 g / L magnesium sulfate, and 0.1 g / L calcium carbonate.

[0049] Fermentation culture conditions: The seed culture was inoculated into a 250ml Erlenmeyer flask containing fermentation culture medium, and the culture temperature was controlled at 30℃ and 37℃ respectively.

[0050] Example 1 Construction of plasmids PKSVT-Δpks5, PKSVT-Δpks6 and PKSVT-Δpks10 Construction of the insert fragment: Genomic DNA was extracted from the host bacillus *Bacillus belye* using a bacterial genomic DNA extraction kit according to the manufacturer's instructions. Using the extracted genomic DNA as a template, appropriate PCR amplification primers were designed, and their sequences are shown in Table 1, where F represents the forward primer and R represents the reverse primer. PCR amplification was performed using primer pairs pks5-up-F / pks5-up-R, pks6-up-F / pks6-up-R, pks10-up-F / pks10-up-R and pks5-down-F / pks5-down-R, pks6-down-F / pks6-down-R, pks10-down-F / pks10-down-R, and pks5-down-F / pks5-down-R, pks6-down-F / pks6-down-R, pks10-down-F / pks10-down-R to obtain upstream and downstream homologous arm fragments. The amplification products were verified by agarose gel electrophoresis and then purified for later use.

[0051] Subsequently, overlapping extension PCR technology was used to connect the upstream and downstream homologous arms through homologous recombination to construct a complete insert fragment, which was then verified by electrophoresis and purified for later use.

[0052] One-step cloning reaction: The obtained insert fragment and the restriction endonuclease-linearized plasmid PKSVT were ligated in vitro using seamless cloning. All ligation products were transformed into competent cells and immediately incubated on ice for 15 min. Following this, the cells were heat-shocked at 42°C for 90 sec, then immediately incubated on ice for 2 min. 900 μL of antibiotic-free LB medium was added to a clean bench, and the cells were incubated on a rotating shaker at 37°C and 220 rpm for 40 min. After incubation, the cells were plated on LB agar plates containing kanamycin (50 μg / mL) and incubated upside down at 37°C for 12 h. Once single colonies emerged, several colonies were selected and PCR was performed using primers Δpks5-F / Δpks5-R, Δpks6-F / Δpks6-R, and Δpks10-F / Δpks10-R. Agarose gel electrophoresis was used to check for the release of bands of the expected size, confirming the correctness and sequence integrity of the insert fragment. The recombinant plasmids PKSVT-Δpks5, PKSVT-Δpks6, and PKSVT-Δpks10, used to knock out the polyketide synthase gene, were finally obtained. The plasmid maps are shown below. Figure 3 .

[0053] Table 4. Primer sequence information table.

[0054]

[0055] Example 2 Construction of recombinant Bacillus belye Construction of Bacillus belye TK1926Δpks: The plasmids PKSVT-Δpks5, PKSVT-Δpks6, and PKSVT-Δpks10 were introduced into *Bacillus belyssae* TK1926 using electroporation, and the target recombinant strain was obtained through kanamycin resistance testing. The specific experimental steps are as follows: (1) Methylation of plasmids: The constructed knockout vector was transformed into EC135 competent cells and cultured at 37°C and 220 rpm. When the OD600 value of the culture medium was 0.2, 80 μL of 50 mg / mL arabinose aqueous solution was added to induce methylation, and the cells were cultured overnight at 30°C in a shaker.

[0056] (2) Preparation and electroporation of competent cells: Culture medium for preparing competent cells of Bacillus belyssus: LBS medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 5 g / L, sorbitol 9.1085 g / L; Resuscitation medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 5 g / L, sorbitol 9.1085 g / L, mannitol 6.92246 g / L.

[0057] Preparation process of Bacillus belysin competent cells: First, single colonies were picked from LB agar plates and inoculated into 5 mL autoclaved LB liquid tubes. These tubes were then incubated at 37°C and 220 rpm for 10 h to allow the cells to reach the logarithmic growth phase. Subsequently, the culture was transferred to a 500 mL shake flask containing 50 mL of LB liquid medium, and the initial OD600 value was adjusted to 0.1. The flasks were then incubated with shaking for another 3 h until the OD600 value reached 0.4–0.6. Competent cells were then prepared according to the methods reported in the literature.

[0058] Electroporation: Approximately 5 μg of methylated plasmid was mixed with competent cells and transferred to an electroporation cuvette. Electroporation was performed at 2.5 kV, 25 μF, and 200 Ω. Immediately after electroporation, 900 mL of preheated electroporation recovery medium (37 °C) was added, and the cells were incubated at 37 °C for 3 h in a shaker to promote cell recovery. After recovery, the cells were centrifuged at 4000 rpm for 5 min, collected, and plated onto LB agar plates containing 50 μg / ml kanamycin. The cells were cultured until monoclonal colonies formed.

[0059] (3) Screening of target strains: single exchange validation First, colony PCR was performed to verify the single colonies grown on the plates. Knockout vector verification primers L1 and L2 were used to confirm the entry of the PKSVT-Δpks plasmid into *Bacillus belyssae*. Subsequently, single colonies that tested positive for colony PCR were picked and inoculated into LB liquid medium containing 50 μg / ml kanamycin. The medium was incubated at 45°C and 220 rpm for 5-6 consecutive passages. After dilution and plating, single colonies were selected for colony PCR verification. Since single crossover can occur between upstream and downstream homologous sequences, two sets of primers were used for verification: pks5-1-F / L2 and pks5-2-R / L1, pks6-1-F / L2 and pks6-2-R / L1, and pks10-1-F / L2 and pks10-2-R / L1. The 1% agarose gel verification pattern is shown below. Figure 4 .

[0060] Double-swap verification Successfully exchanged single colonies were selected and inoculated into antibiotic-free LB broth medium. The culture was incubated at 37°C and 220 rpm for 12 h, and passaged 3-4 times. The culture was then diluted and spread onto antibiotic-free LB plates, and cultured until single colonies formed. Finally, single colonies were selected for colony PCR verification using primers pks5-1-F / pks5-2-R, pks6-1-F / pks6-2-R, and pks10-1-F / pks10-2-R. The PCR products were sent to a biotechnology company for sequencing analysis. The nucleic acid gel verification pattern is shown below. Figure 3 To further confirm the correct recombination of the genome, the following results were obtained: Bacillus belyssus PKSVT-Δpks5, PKSVT-Δpks6, and PKSVT-Δpks10.

[0061] (4) The OD600 of the knockout bacteria was measured in LB medium by shaking flask culture (37℃, 220rpm), and the growth curve was plotted as follows: Figure 5 .

[0062] Example 3 Penicillium antibacterial experiment (1) Preparation of Penicillium suspension 100 μL of Penicillium mold stored at -80°C was taken out and inoculated onto a PDA solid medium plate using a dilution spread method. The plate was then incubated at 35°C for 7 days (until spore concentration reached over 85%). The plate was washed with ddH2O to remove spores, and the spore suspension was filtered through 8 layers of sterile gauze to remove vegetative mycelium. The spore suspension was collected and poured into a 50 mL centrifuge tube. An appropriate amount of ddH2O was added, and the spores were counted under a microscope using a hemocytometer to prepare a Penicillium mold suspension (1.0 × 10⁻⁶). -6 (spores / mL), store in a 4°C refrigerator for later use.

[0063] (2) Preparation of double discs Melt the plain agar medium by heating and pour it into sterile empty glass petri dishes (approximately 10 mL per dish). Place the dishes on a laminar flow hood to allow them to solidify. Add 1% Penicillium suspension to PDA medium (pH 3.5±0.1) cooled to 50°C and mix gently. Then, use a pipette to add 10 mL of PDA medium containing the bacterial suspension to the previously solidified plain agar medium. After it cools and solidifies, seal the container with film and store it in a 4°C refrigerator for later use.

[0064] (3) Preparation of antibacterial plates A four-point confrontation culture method was used. Four Oxford cups were symmetrically placed 2.5 cm from the center of a double-plate culture. 100 μL of fermentation broth obtained by centrifugation (12000 rpm, 5 min) was added to each Oxford cup. After standing at 4℃ for 4 h, the cups were placed in a 35℃ incubator for 48 h to observe the antibacterial effect. The original strain fermentation broth was used as a control. Each treatment was repeated three times, and the experiment was repeated twice. The inhibition rate was calculated using the formula below.

[0065] The antibacterial rate is calculated using the following formula: Inhibition rate (%) = ((A1-A2) / A1)×100% Where: A1 is the colony diameter of the original bacterial control group, mm; A2 is the colony diameter of the recombinant bacterial treatment group, mm.

[0066] Example 4 Fermentation medium optimization Culture media are essential nutrient substrates for the growth, reproduction, and synthesis of various metabolites of microorganisms. The components of the culture medium can significantly regulate the growth rate of microorganisms, the selectivity of metabolic pathways, and the yield of target metabolites. Therefore, this invention systematically optimizes the composition and culture conditions of fermentation culture media.

[0067] 1. Screening of culture medium formulations Three different fermentation media with the following components were designed and formulated for screening: Culture medium A: glucose 10 g / L, yeast extract 10 g / L, potassium dihydrogen phosphate 0.5 g / L, manganese sulfate 0.005 g / L, magnesium sulfate 0.05 g / L, calcium carbonate 0.1 g / L, pH 7.6.

[0068] Culture medium B: sucrose 10 g / L, beef extract 10 g / L, magnesium sulfate 0.001 g / L, pH 6.0.

[0069] Culture medium C: corn starch 10 g / L, yeast extract 25.1 g / L, manganese sulfate 0.0051 g / L, potassium dihydrogen phosphate 0.5 g / L, pH 7.5.

[0070] 2. Optimization of culture temperature After adjusting the initial pH of each of the above culture media to the specified value, a 2% inoculum of *Bacillus belyssima* engineered strain seed culture was inoculated, and shake-flask fermentation was carried out at 30℃ and 37℃, respectively, for a fermentation period of 48 hours. After fermentation, the fermentation supernatant was collected by centrifugation, and its inhibitory activity against *Penicillium* was determined using the plate inhibition zone method, and the relative inhibition rate was calculated.

[0071] 3. Optimization Results Experimental results are as follows Figures 6-8 As shown, when using culture medium C (formulation: 10 g / L corn starch, 25.1 g / L yeast extract, 0.0051 g / L manganese sulfate, 0.5 g / L potassium dihydrogen phosphate) and controlling the fermentation conditions at 37℃ and pH 7.5, the engineered strain synthesized the highest yield of anti-Penicillium active substances, and the antibacterial rate of the fermentation product reached its peak, which was significantly better than other culture medium formulations and 30℃ culture conditions.

[0072] 4. Determining the optimal process Based on the above screening results, the present invention determines that the preferred fermentation medium for producing anti-Penicillium active substances comprises: corn starch, yeast extract, manganese sulfate, and potassium dihydrogen phosphate, with the preferred concentration ratio being: corn starch 10 g / L, yeast extract 25.1 g / L, manganese sulfate 0.005 g / L, and potassium dihydrogen phosphate 0.5 g / L. The optimal fermentation conditions are: a culture temperature of 37℃ and a pH value maintained at 7.5. Under this optimized process, fermentation products with strong antibacterial activity can be produced stably and efficiently.

[0073] In summary, the optimal culture medium formulation and fermentation conditions for the subsequent fermentation system were finally determined, and the results are as follows: Figure 8 As shown, in the optimized fermentation medium, the average diameter of the inhibition zone of the original strain was 22.1 mm, and the average diameter of the inhibition zone of the ΔPKS6 strain was 24.3 mm. Therefore, the inhibition rate of the optimized recombinant strain ΔPKS6 fermentation broth increased by 10.4% compared with that of the original strain fermentation broth.

[0074] This invention successfully constructed three engineered strains of *Bacillus belye* with simplified PKS gene clusters, and significantly improved the yield of anti-Penicillium active substances in the ΔPKS6 strain through fermentation process optimization. The methods and results of this invention provide a new technical approach for green prevention and control of agricultural diseases and biological preservation of food. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of this invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

[0075] For any points not covered above, existing technologies shall apply.

[0076] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A genetically engineered bacterium resistant to Penicillium berberis, characterized in that, A cluster of polyketide synthase genes in the genome of the *Bacillus belyssae* was knocked out or inactivated, and the gene cluster included one of the PKS5, PKS6, and PKS10 gene clusters.

2. The genetically engineered strain of *Bacillus bellsii* resistant to *Penicillium* according to claim 1, characterized in that, The gene cluster that was knocked out or inactivated is PKS6.

3. The genetically engineered strain of *Bacillus bellsii* resistant to *Penicillium* according to claim 1, characterized in that... The preservation number of the Bacillus belyssus is CGMCC No. 36999.

4. The genetically engineered strain of *Bacillus bellsii* resistant to *Penicillium* according to claim 3, characterized in that... The pks5 gene cluster is located at positions 1426080-1514310 of the whole genome sequence, the pks6 gene cluster is located at positions 1735334-1845433 of the whole genome sequence, and the pks10 gene is located at positions 2475831-2496721 of the whole genome sequence, based on the whole genome sequence position of Bacillus belyss with the GenBank accession number CGMCC No. 36999.

5. The method for constructing the genetically engineered strain resistant to *Bacillus berberis* as described in claim 4, characterized in that, Includes the following steps: Provide the Bacillus belesiensis as described in claim 3; Gene manipulation elements were designed to knock out or inactivate a polyketide synthase gene cluster in the genome of the Bacillus belyssus. The gene manipulation element is introduced into the Bacillus belyssus, causing the polyketide synthase gene cluster to be knocked out or inactivated. Obtain Bacillus belyss genetically engineered bacteria in which the polyketide synthase gene cluster has been knocked out or inactivated.

6. The construction method according to claim 5, characterized in that, The gene manipulation elements include homologous recombination fragments, CRISPR / Cas gene editing systems, transposon elements, or combinations thereof.

7. The construction method according to claim 5, characterized in that, The polyketide synthase gene cluster is one of the PKS5, PKS6, and PKS10 gene clusters.

8. A fermentation method for the genetically engineered Bacillus venereum resistant to Penicillium berberis as described in any one of claims 1-4, characterized in that, The culture medium is inoculated into the yeast extract for fermentation to obtain the target fermentation product. The culture medium is formulated as follows: corn starch 8~12 g / L, yeast extract 20~30 g / L, manganese sulfate 0.001~0.01 g / L, potassium dihydrogen phosphate 0.3~0.7 g / L, pH 7.0~8.

0.

9. The fermentation method according to claim 8, characterized in that, The inoculation amount is 2%~5% of the culture medium, and fermentation is carried out at 30℃~37℃ for 48~72h.

10. Use of the genetically engineered Bacillus belye strain according to any one of claims 1-4 or the fermentation product obtained by the fermentation method according to any one of claims 8-9 in the preparation of a microbial preparation for inhibiting the growth of Penicillium.

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