Streptomyces hygroscopicus engineering strain and construction and application thereof

Abstract

The invention provides a streptomyces hygroscopicus engineering strain as well as construction and application thereof, and relates to the technical field of biology, the engineering strain takes streptomyces hygroscopicus CL-1 as a starting host and is obtained by heterologous expression of an epsilon-polylysine synthetase encoding gene pls, and the nucleotide sequence of the epsilon-polylysine synthetase encoding gene pls is shown as SEQ ID NO.1. Streptomyces hygroscopicus CL-1 is used as a host, the epsilon-polylysine synthetase coding gene derived from streptomyces albus is subjected to heterologous expression, the obtained recombinant strain has high epsilon-polylysine production capacity, and under the optimized fermentation condition, the yield of epsilon-polylysine is 0.87 g / L, and industrial scale production requirements are met. Meanwhile, the broad-spectrum antibacterial activity to various plant pathogenic fungi and common bacteria, including colletotrichum gloeosporioides, fusarium oxysporum, phytophthora capsici and the like, is also remarkably improved. Therefore, the streptomyces hygroscopicus has important application in the aspects of production of epsilon-polylysine, prevention and control of pathogenic microorganisms, preservation and the like, and the industrial and agricultural utilization potential of the streptomyces hygroscopicus is expanded.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to an engineered strain of Streptomyces hygroscopicus and its construction and application. Background Art

[0002] Streptomyces albus is a species of the genus Streptomyces. This species can produce ε-polylysine, which is a homopolymer formed by the dehydration condensation of 25 to 35 L-lysine residues between α-carboxyl groups (α-COOH) and ε-amino groups (ε-NH-). Its relative molecular mass is generally between 2500 and 4500 Da. ε-Polylysine is mainly synthesized by Streptomyces through extracellular secretion and is a natural antimicrobial peptide derived from microorganisms. As a new green antimicrobial agent, ε-polylysine has excellent properties such as broad-spectrum antimicrobial activity, safety and non-toxicity, good biocompatibility, strong biodegradability, good water solubility and high thermal stability. It is widely used in food preservation, biomedicine and agricultural disease prevention and control. In recent years, research has shown that ε-polylysine not only inhibits bacteria but also exhibits significant inhibitory activity against a variety of plant pathogenic fungi, garnering increasing attention for its application in agricultural disease control. For example, ε-polylysine can effectively prevent postharvest fungal infections in apples, citrus fruits, red dates, kiwifruit, and various vegetables, reducing losses caused by fungal decay. However, the strain's relatively weak metabolic capacity and environmental adaptability limit its application.

[0003] Streptomyces hygroscopicus is a Streptomyces species with strong metabolic activity and strong environmental adaptability. It can survive at low temperatures and under various culture conditions and has good industrial application potential. For example, the Chinese invention patent publication number CN118207136A discloses the CL-1 strain of Streptomyces hygroscopicus, which has been shown to prevent and treat a variety of plant diseases including Colletotrichum gloeosporioides, Fusarium oxysporum, Phytophthora capsici, Rhizoctonia solani, and Gibberella graminis, showing good application prospects. However, this strain of Streptomyces hygroscopicus cannot produce ε-polylysine. Therefore, its scope of application and control effect are limited.

[0004] Therefore, it is necessary to develop an engineered strain to overcome the shortcomings of the above two bacteria in order to improve the disease control effect and the application scope and potential in biological agricultural prevention and preservation. Summary of the Invention

[0005] The object of the present invention is to solve the above-mentioned technical problems and provide an engineered strain of Streptomyces hygroscopicus, which can ferment and produce high yields of ε-polylysine and increase the scope and potential of its application in biological agricultural prevention and control and preservation. Another object of the present invention is to provide a method for constructing the strain. A further object of the present invention is to provide the use of the strain in the production of ε-polylysine, drugs for preventing and controlling biological diseases, or preservatives for preventing and controlling spoilage.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] An engineered strain of Streptomyces hygroscopicus is obtained by heterologously expressing an ε-polylysine synthase encoding gene pls using Streptomyces hygroscopicus var. angustmyceticus CL-1 as a starting host. The nucleotide sequence of the ε-polylysine synthase encoding gene pls is shown in SEQ ID NO.1.

[0008] The present invention uses Streptomyces hygroscopicus CL-1 as a host to heterologously express the gene encoding ε-polylysine synthase (pls) from Streptomyces albus. The resulting recombinant strain not only has a strong ability to produce ε-polylysine, but also exhibits broad-spectrum antibacterial activity against a variety of plant pathogenic fungi and common bacteria, including Colletotrichum gloeosporioides, Fusarium oxysporum, and Phytophthora capsici. Therefore, it has important applications in ε-polylysine production and pathogen control, expanding the industrial and agricultural utilization potential of Streptomyces hygroscopicus.

[0009] Among them, the Streptomyces hygroscopicus var. angustmyceticus CL-1 is obtained by screening from lake sediments; specifically, the detailed information of the Streptomyces hygroscopicus CL-1 has been disclosed in patent CN118207136A, and the deposit number is CCTCC M 20231221.

[0010] Furthermore, the promoter sequence of the ε-polylysine synthase encoding gene pls heterologously expressed by the Streptomyces hygroscopicus engineered strain is the nucleotide sequence shown in any one of SEQ ID NOs. 2 to 4.

[0011] The present invention found that by optimizing the promoter sequence and implementing an adaptive evolution strategy, the yield of ε-polylysine can be significantly improved, and ultimately 0.87 g / L of the target product is obtained during the fermentation process.

[0012] Furthermore, the engineered strain of Streptomyces hygroscopicus uses the pSET152 vector plasmid as an expression vector.

[0013] The present invention provides a method for constructing the above-mentioned Streptomyces hygroscopicus engineered strain, characterized in that it comprises the following steps:

[0014] S1. The ε-polylysine synthetase encoding gene pls was cloned into the expression vector pSET152 containing any promoter sequence as shown in SEQ ID NO.2-4 to obtain a recombinant plasmid;

[0015] S2. Transforming the recombinant plasmid described in step S1 into competent cells of the donor bacterium E. coli ET12567 to construct a recombinant donor bacterium;

[0016] S3. The recombinant donor bacteria described in step S2 are mixed with spores of Streptomyces hygroscopicus CL-1 for conjugation transfer to construct the engineered strain of Streptomyces hygroscopicus.

[0017] The present invention provides the use of the above-mentioned Streptomyces hygroscopicus engineered strain or the construction method of the above-mentioned Streptomyces hygroscopicus engineered strain as or in the preparation of a medicine for preventing and treating biological diseases or a preservative for preventing and treating spoilage.

[0018] Preferably, the biological disease or spoilage is caused by bacterial, fungal pathogens or a combination thereof.

[0019] More preferably, the biological disease or spoilage is caused by one or any combination of pathogenic fungi including Colletotrichum gloeosporioides, Fusarium oxysporum, and Phytophthora capsici.

[0020] The present invention provides the application of the above-mentioned Streptomyces hygroscopicus engineered strain or the construction method of the above-mentioned Streptomyces hygroscopicus engineered strain in the production of ε-polylysine.

[0021] Furthermore, before fermentation to produce ε-polylysine, the engineered strain of Streptomyces hygroscopicus is subjected to adaptive evolution, wherein the engineered strain of Streptomyces hygroscopicus is subcultured every 24 to 48 hours in TSB fermentation medium containing 0.5-3 g / L ε-polylysine for a total of 30 to 60 days.

[0022] The present invention provides a medicine for preventing and treating biological diseases or a preservative for preventing and treating spoilage. The medicine for preventing and treating biological diseases or the preservative for preventing and treating spoilage contains the above-mentioned engineered strain of Streptomyces hygroscopicus or its fermentation product.

[0023] Furthermore, the fermented product is prepared by the following method:

[0024] P1. After activating the engineered strain of Streptomyces hygroscopicus, inoculate it into a seed culture medium for cultivation to obtain a seed solution containing genetically recombinant bacteria. The seed solution OD 600 is 4.5-8.5; preferably, the seed culture medium is M3G or TSB medium;

[0025] P2. The seed solution obtained in step P1 was inoculated into the fermentation medium at an inoculum size of 2% to 12% for fermentation in a fermenter;

[0026] Preferably, the fermentation tank is fermented at a temperature of 28-37° C. and a shaking speed of 200 rpm for 48-96 hours to obtain a fermented product.

[0027] Furthermore, the fermentation medium formula is as follows:

[0028] Glucose 30-80g / L, soybean powder 5-15g / L, (NH4)2SO4 5-15g / L, yeast powder 2-8g / L, K2HPO4 0.5-1.0g / L, KH2PO4 0.5-1.5g / L, MgSO4·7H2O 0.4-0.8g / L, FeSO4·7H2O 0.02-0.04g / L, ZnSO4·7H2O 0.02-0.06g / L.

[0029] The beneficial effects of the present invention are as follows:

[0030] (1) The present invention uses Streptomyces hygroscopicus CL-1 as a host for the first time, and constructs a recombinant strain capable of synthesizing ε-polylysine by heterologously expressing the ε-polylysine synthase gene (pls) from Streptomyces albus. The ε-polylysine expression efficiency is high, the yield is significantly improved, and the strain is stable. Furthermore, by optimizing the promoter of the ε-polylysine synthase encoding gene pls and combining it with an adaptive evolution strategy, the expression efficiency of the target gene in Streptomyces hygroscopicus is significantly improved, and the tolerance and genetic stability of the recombinant strain to ε-polylysine are enhanced. Under optimized fermentation conditions, the yield of ε-polylysine is 0.87 g / L, showing good yield level and stability, meeting the needs of industrial-scale production.

[0031] (2) The engineered strain of Streptomyces hygroscopicus of the present invention has broad-spectrum and more efficient antibacterial activity: the supernatant of the fermentation broth of the constructed recombinant bacteria shows significantly improved inhibitory activity against a variety of pathogenic fungi, especially against Colletotrichum gloeosporioides, Fusarium oxysporum and Phytophthora capsici, and has a broad-spectrum, efficient and safe biological control potential.

[0032] (3) The recombinant bacteria constructed in the present invention provide a new production approach and technical support for its efficient application in multiple fields such as food preservation, medical preservation and agricultural disease prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the PCR verification result of the recombinant strain of the present invention.

[0034] Figure 2Production of ε-polylysine by recombinant strains containing different promoters.

[0035] Figure 3 MALDI-TOF-MS analysis results of the fermentation broth of the recombinant strain.

[0036] Figure 4 The production of ε-polylysine synthesized by the recombinant strain before and after adaptive evolution. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0038] The experiments, detection and preparation processes involved in the present invention all adopt conventional techniques in related fields such as molecular biology and biochemistry, and these methods have been disclosed in detail in existing literature.

[0039] In the following examples, the reagents and culture medium used are as follows:

[0040] (1) Liquid LB medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride; solid medium supplemented with 2% agar powder.

[0041] (2) ISP2 solid culture medium: glucose 4 g / L, yeast powder 4 g / L, maltose extract 4 g / L powder, agar powder 20 g / L, adjust pH to 7.0.

[0042] (3) MS solid medium: 20 g / L soybean powder, 20 g / L mannitol, 20 g / L agar powder, adjust the pH to 7.2, and add 10 mM MgCl2 before pouring the plate.

[0043] (4) Fermentation medium: glucose 50 g / L, soybean powder 10 g / L, (NH4)2SO4 10 g / L, yeast powder 5 g / L, K2HPO4 0.8 g / L, KH2PO4 1.39 g / L, MgSO4·7H2O 0.5 g / L, FeSO4·7H2O 0.03 g / L, ZnSO4·7H2O 0.04 g / L, ammonia water to adjust the pH to 6.86.

[0044] In the following examples, the method for detecting the yield of ε-polylysine is as follows:

[0045] The Itzhaki method was used for detection. The specific operation steps were referred to the literature (Itzhaki RF. Colorimetric method for estimating polylysine and polyarginine. Anal Biochem, 1972, 50: 569-574).

[0046] Example 1 Construction of recombinant Streptococcus hygroscopicus

[0047] This example provides an engineered strain of Streptomyces hygroscopicus, obtained by heterologously expressing the gene encoding ε-polylysine synthase, pls, using Streptomyces hygroscopicus var. angustmyceticus CL-1 as a starting host. The nucleotide sequence of the gene encoding ε-polylysine synthase, pls, is shown in SEQ ID NO. 1. The specific experimental procedure is as follows:

[0048] 1. Experimental Methods

[0049] (1) Obtaining the gene encoding ε-polylysine synthase

[0050] The nucleotide sequence of the ε-polylysine synthase gene in the prior art is shown in GenBank No.: JF427577.1 (derived from Streptomyces albulus PD-1). Analysis shows that the GC content of the ε-polylysine synthase encoding gene is 76%; the nucleotide sequence of the existing ε-polylysine synthase encoding gene is shown in SEQ ID NO.1.

[0051] (2) Construction of recombinant plasmid

[0052] First, the nucleotide sequence of the ε-polylysine synthase gene (SEQ ID NO. 1) was amplified by PCR using the primer sequences listed in Table 1. Subsequently, the amplified products were assembled into the linearized vector of the integrative plasmid pSET152, which contains the strong promoters ermEp*, kasOp*, and SP44, using a one-step cloning kit, to construct recombinant plasmids expressing three different promoters.

[0053] The ligation reaction product was transformed into E. coli DH5α competent cells and plated onto LB solid medium plates containing 50 μg / mL apramycin sulfate. After incubation at 37°C for approximately 12 hours, single colonies were selected for PCR verification. Electrophoresis confirmed the correct recombinant strain, and further samples were sent for sequencing to obtain the correct recombinant transformant.

[0054] Wherein, the nucleotide sequences of the strong promoters ermEp*, kasOp*, and SP44 are shown in SEQ ID NO.2-4.

[0055] The PCR amplification system is as follows: forward primer F 2 μL, reverse primer R 2 μL, PUC19-pls template 2 μL, 2×Phanta Max Master Mix 25 μL, and ddH2O 19 μL; the PCR reaction procedure is as follows: pre-denaturation at 95°C for 5 min, 30 cycles of 95°C for 30 s, 60°C for 15 s, and 72°C for 180 s, and full extension at 72°C for 5 min.

[0056] Among them, the linearized integration plasmid pSET152 vector containing the strong promoters ermEp*, kasOp*, and SP44 has a reaction system of: 30 μL of plasmid, 5 μL of SwiftCut NdeⅠ, 5 μL of SwiftCut XbaⅠ, 5 μL of 10×SwiftCut Color Buffer, and 5 μL of ddH2O, and incubated at 37°C for 30 min.

[0057] Table 1 Primer sequences used

[0058] Primer name Primer sequence (5'-3') Primer number ermEp*-pls-F ggttggtaggatccacatatgATGTCGTCGCCCCTTCTCG SEQ ID NO.5 ermEp*-pls-R ccgcggatcctctagtctagaTCACGCGGCCGCACCTCC SEQ ID NO.6 kasOp*-pls-F aggactggggggagttcatatgATGTCGTCGCCCCTTCTCG SEQ ID NO.7 kasOp*-pls-R cgcggccgcggatcctctagaTCACGCGGCCGCACCTCC SEQ ID NO.8 SP44-pls-F taagtaaggagtgtccatatgATGTCGTCGCCCCTTCTCG SEQ ID NO.9 SP44-pls-R cgcggccgcggatcctctagaTCACGCGGCCGCACCTCC SEQ ID NO.10

[0059] (3) Construction of recombinant strains

[0060] Gently mix the prepared plasmid DNA with 100 μL of competent E. coli ET12567, let it stand in an ice bath for 30 minutes, then immediately place it in a 42°C water bath for a heat shock treatment for 90 seconds, and then quickly transfer it to an ice bath to cool for 2 minutes. Then add approximately 600 μL of liquid LB culture medium and incubate it at 37°C and 200 rpm for 60 minutes to restore bacterial activity. After the above incubation is completed, centrifuge it at 5000 rpm for 3 minutes, discard the supernatant liquid, and retain approximately 200 μL of liquid. Remix it and evenly spread it on an LB agar plate containing 50 μg / mL apramycin sulfate, 25 μg / mL kanamycin, and 25 μg / mL chloramphenicol antibiotics. Incubate until a single colony is formed.

[0061] Randomly select 3 to 4 individual colonies and inoculate them into 20 mL of LB liquid medium containing the same antibiotic combination as above (37°C, 200 rpm, 12 h). After incubation, centrifuge at 5000 rpm for 5 min to remove the supernatant. Wash the cells twice with antibiotic-free LB medium and resuspend them in 2 mL of antibiotic-free LB medium to obtain the recombinant donor bacterial suspension.

[0062] The prepared recombinant donor bacterial suspension (500 μL) was then mixed evenly with 200 μL of hygroscopic Streptomyces CL-spore suspension, and 100 μL, 200 μL, and 400 μL of the bacterial solution were respectively applied to the surface of three different MS solid culture media and cultured at a constant temperature of 30°C for 24 hours. After 24 hours, 1 mL of sterile water containing 1 mg of apramycin sulfate and 1 mg of nalidixic acid was added to each plate surface and covered. After another 3 to 4 days of culture, the transferred strain (conjugative transfer product) was obtained. The cultured conjugative transfer product was picked and inoculated into TSB liquid culture medium and cultured in a shaking incubator at 30°C for 48 hours (200 rpm). After the culture was completed, part of the bacterial solution was stored in glycerol for a long time, and the genomic DNA of the remaining bacterial solution was extracted and PCR verification was performed.

[0063] 2. Experimental Results

[0064] PCR test results can be found in Figure 1 Lane M is a 15,000 bp marker; lanes 1-3 are recombinant strains carrying different promoters, and the amplification results of their heterologously expressed pls genes were verified by PCR.

[0065] Through the above steps, the following three recombinant strains were successfully obtained: CL-1 / pSET15-ermEp*-pls, CL-1 / pSET15-kasOp*-pls, and CL-1 / pSET15-SP44-pls. The spore suspension of Streptomyces hygroscopicus CL-1 was collected after 10 days of culture on ISP2 solid medium.

[0066] Example 2 Effects of different promoters on the production of ε-polylysine by recombinant bacteria

[0067] 1. Experimental Methods

[0068] To evaluate the effects of different promoters on the ε-polylysine biosynthesis capacity of recombinant strains, recombinant strains containing different promoters were selected and their ε-polylysine production was evaluated to determine the optimal promoter. Several recombinant strains were inoculated into fermentation medium and cultured in shake flasks at 30°C and 200 rpm for 72 hours. After fermentation, the fermentation broth was centrifuged and ε-polylysine production was determined using the methyl orange colorimetric assay.

[0069] The fermentation medium formula is:

[0070] Glucose 50g / L, soybean powder 10g / L, (NH4)2SO4 10g / L, yeast powder 5g / L, K2HPO4 0.8g / L, KH2PO4 1.39g / L, MgSO4·7H2O 0.5g / L, FeSO4·7H2O 0.03g / L, ZnSO4·7H2O 0.04g / L, ammonia water to adjust pH to 6.86.

[0071] 2. Experimental Results

[0072] like Figure 2 As shown in the figure, experimental results showed that the production of ε-polylysine increased with increasing promoter strength. Among them, the recombinant strain CL-1-SP44-pls, constructed using the strong promoter SP44, performed best, with an ε-polylysine production of 0.41 g / L and a dry cell weight (DCW) of 4.22 g / L, both higher than those of other recombinant strains. This increase in production was primarily attributed to the high expression activity of the SP44 promoter. According to literature reports, the strength of the SP44 promoter is approximately 36.82 times that of the ermEp* promoter. In summary, the SP44 promoter is the most effective in driving the expression of the gene encoding ε-polylysine synthase. In subsequent experiments, the recombinant Streptomyces hygroscopicus CL-1 / pSET152-SP44-pls was selected as the expression strain.

[0073] Example 3 Matrix-assisted laser desorption ionization time-of-flight mass spectrometry determination of components of the fermentation broth of the recombinant strain

[0074] 1. Experimental Methods

[0075] This example aims to characterize the molecular weight distribution of the products synthesized in the fermentation broth of the recombinant strain and verify its ability to synthesize ε-polylysine. The recombinant Streptomyces hygroscopicus CL-1 / pSET15-SP44-pls strain screened in Example 2 was selected as the starting strain and fermented in shake flasks. After fermentation, the fermentation supernatant was pretreated: the pH of the filtrate was first adjusted to 3.0 with hydrochloric acid and cooled at 4°C. A methanol / acetone (3:1 volume ratio) organic solvent mixture was then added. The amount of the organic reagent mixture was adjusted to account for 67% of the total volume of the mixture. The mixture was then allowed to stand at 4°C overnight to induce precipitation of the target product. The precipitate was collected by low-speed centrifugation and reconstituted with deionized water. The resulting solution was then used for subsequent mass spectrometry analysis. Molecular weight analysis was performed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS). The mass spectrometry test was performed in the positive ion mode. The matrix used was 2,5-dihydroxybenzoic acid. The sample and the matrix were mixed in an appropriate ratio and spotted on a carrier plate. After drying, the detection was performed.

[0076] 2. Experimental Results

[0077] The test results are as follows Figure 3As shown, the relative molecular mass distribution of the fermentation product of the recombinant strain ranged from 3477 to 4502 Da, with the signal mainly concentrated near a molecular weight of 4118 Da, indicating that the recombinant engineered strain can stably synthesize ε-polylysine of a certain chain length. This result further verified that the recombinant strain obtained through promoter optimization has the ability to efficiently convert L-lysine to ε-polylysine, providing a foundation for subsequent production.

[0078] Example 4 Adaptive evolution improves the tolerance of recombinant strains to ε-polylysine

[0079] 1. Experimental Methods

[0080] This example aims to improve the tolerance and production capacity of recombinant strains for ε-polylysine through adaptive evolution. The recombinant Streptomyces hygroscopicus CL-1 / pSET15-SP44-pls obtained in Example 2 was selected as the starting strain and adaptively evolved in TSB (Tryptone Soy Broth) liquid culture medium containing different concentrations of ε-polylysine (0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, and 3.0 g / L, respectively). The specific evolution process is as follows: the initial starting strain is inoculated into TSB medium containing 0.5 g / L ε-polylysine, and subculture is performed every 24 hours. Each time, 10% (v / v) of the culture volume is transferred to fresh medium containing the same concentration of ε-polylysine for a total of six consecutive subcultures. The strain obtained at this stage was then transferred to a medium containing 1.0 g / L ε-polylysine and subcultured for six generations in the same manner, gradually increasing the ε-polylysine concentration until six generations of subculture were completed under different ε-polylysine concentrations. Finally, after 30 generations of continuous adaptive evolution, an evolved recombinant strain tolerant to high ε-polylysine concentrations was obtained.

[0081] To verify the effectiveness of adaptive evolution, a shake flask fermentation experiment was conducted using a control strain that had not undergone adaptive evolution. The experimental conditions were: 30°C, 200 rpm shaker speed, and 72 hours of fermentation. After fermentation, the yield of ε-polylysine in the fermentation broth was accurately measured.

[0082] 2. Experimental Results

[0083] Specific experimental results such as Figure 4The results showed that the recombinant strain after adaptive evolution exhibited significantly enhanced ε-polylysine production capacity, with ε-polylysine yield and DCW reaching 0.87 g / L and 6.79 g / L, respectively. Compared with the unevolved control strain (0.43 g / L and 4.25 g / L, respectively), these increases were 102.33% and 59.76%, respectively. This indicates that the adaptive evolution strategy of gradually increasing the ε-polylysine concentration in the culture medium can effectively improve the tolerance and production performance of the recombinant strain, providing an effective new strategy for the industrial and efficient production of ε-polylysine.

[0084] Example 5 Evaluation of the inhibitory activity of the fermentation broth of recombinant Streptomyces hygroscopicus CL-1 / pSET15-SP44-pls against pathogenic fungi

[0085] 1. Experimental Methods

[0086] The inhibitory activity of recombinant Streptomyces hygroscopicus CL-1 / pSET15-SP44-pls against plant pathogenic fungi was determined using a mycelial growth rate assay. Both Streptomyces hygroscopicus CL-1 and the recombinant strain CL-1 / pSET15-SP44-pls were fermented and cultured according to the conditions described in Example 1: inoculated into the same culture medium and cultured with shaking at 30°C and 200 rpm for 72 hours. After fermentation, the fermentation broth was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected and sterilized by filtration through a 0.22 μm filter. The ε-polylysine concentration in the fermentation broth of the recombinant strain CL-1 / pSET15-SP44-pls was controlled at 50 mg / L. 1 mL of sterile fermentation supernatant or treated solution was thoroughly mixed with 19 mL of PDA medium cooled to approximately 55°C and poured onto a plate to prepare a solid culture medium. Four treatments were set up in the experiment: ① a control group without any fermentation products; ② a control group supplemented with fermentation supernatant of the CL-1 strain; ③ a control group supplemented with fermentation supernatant of the recombinant strain CL-1 / pSET15-SP44-pls (containing 50 mg / L ε-polylysine); and ④ a 50 mg / L ε-polylysine solution exogenously added to the basal PDA medium. Each treatment was replicated three times. A 0.5 cm diameter cake of plant pathogenic fungi was inoculated in the center of each plate (mycelium facing down and attached to the culture medium surface). After incubation, colony diameters were measured using the cross-hatch method, and the relative inhibition rate was calculated according to the following formula: Relative inhibition rate (%) = [(control colony growth diameter - treated colony growth diameter) / (control colony growth diameter - cake diameter)] × 100%.

[0087] 2. Experimental Results

[0088] In this embodiment, the antifungal activity of the fermentation broth of the recombinant strain CL-1 / pSET15-SP44-pls was compared with that of the original strain CL-1, the exogenously added ε-polylysine solution, and the sterile fermentation medium (control group). The results are shown in Table 2. As can be seen from the table, the original strain CL-1 exhibited strong natural antifungal activity against three common plant pathogenic fungi (Colletotrichum gloeosporioides, Fusarium oxysporum, and Phytophthora capsici), with inhibition rates of 78.60%, 70.52%, and 68.53%, respectively. When 50 mg / L ε-polylysine solution was added alone, only 7.53% to 10.26% inhibition rate was shown, indicating that under the experimental conditions, ε-polylysine itself had limited inhibitory effect on pathogens. However, the antifungal rate of the fermentation broth of the recombinant strain CL-1 / pSET15-SP44-pls was significantly improved, reaching 98.62%, 89.68%, and 86.03%, respectively. Compared with the original strain, its antibacterial activity increased by 20.02%, 19.16%, and 17.50%. More importantly, the antibacterial effect of the recombinant bacterial fermentation broth was much higher than the simple physical mixing effect of "using the original bacterial fermentation broth alone + adding an equal amount of ε-polylysine", indicating that the heterologous expression product may exist in a more stable, synergistic, or enhanced form in the bacterial metabolic environment, thereby generating a "1+1>2" technical gain. This result further verifies that recombinant expression of ε-polylysine not only improves product stability and bioavailability, but may also activate or cooperate with other host metabolites to produce new antibacterial mechanisms, which has significant technical advantages and application potential. In summary, the fermentation broth of recombinant Streptomyces hygroscopicus CL-1 / pSET15-SP44-pls has superior antifungal activity compared with the fermentation product alone or exogenously added, and has important application value, providing a solid basis for its further development as a biopesticide or plant protection agent. At the same time, this antibacterial effect can also be applied to the accelerated spoilage caused by the growth of the above-mentioned pathogens on the surface of fruits and vegetables, and can be used as a drug for preventing and controlling biological diseases or a preservative for preventing and controlling spoilage.

[0089] Table 2 Inhibitory effects of fermentation broths of strains CL-1 and CL-1 / pSET15-SP44-pls on different pathogenic fungi

[0090]

[0091] Note: Antibacterial rate a Represents the inhibition rate of Streptomyces hygroscopicus CL-1 fermentation broth; inhibition rate b Representative is exogenous addition of 50mg / Lε-polylysine; inhibition rate c The inhibition rate of Streptomyces hygroscopicus CL-1 / pSET15-SP44-pls fermentation broth (heterologously expressing ε-polylysine synthase), in which the ε-polylysine concentration was controlled at 50 mg / L;

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the solution. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand and can modify or replace the technical solution of the present invention based on the understanding of this solution without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. An engineered strain of Streptomyces hygroscopicus, characterized in that The engineered strain of Streptomyces hygroscopicus is obtained by heterologously expressing the ε-polylysine synthase encoding gene pls using Streptomyces hygroscopicus var. angustmyceticus CL-1 as a starting host. The nucleotide sequence of the ε-polylysine synthase encoding gene pls is shown in SEQ ID NO.

1.

2. The engineered strain of Streptomyces hygroscopicus according to claim 1, characterized in that The promoter sequence of the ε-polylysine synthase encoding gene pls heterologously expressed by the Streptomyces hygroscopicus engineered strain is the nucleotide sequence shown in any one of SEQ ID NOs. 2 to 4.

3. The engineered strain of Streptomyces hygroscopicus according to claims 1 to 2, characterized in that The expression vector of the Streptomyces hygroscopicus engineering strain is a pSET152 vector.

4. The method for constructing an engineered strain of Streptomyces hygroscopicus according to any one of claims 1 to 3, characterized in that: The steps include: S1. The ε-polylysine synthetase encoding gene pls was cloned into the expression vector pSET152 containing any promoter sequence as shown in SEQ ID NO.2-4 to obtain a recombinant plasmid; S2. Transforming the recombinant plasmid described in step S1 into competent cells of the donor bacterium E. coli ET12567 to construct a recombinant donor bacterium; S3. The recombinant donor bacteria described in step S2 are mixed with spores of Streptomyces hygroscopicus CL-1 for conjugation transfer to construct the engineered strain of Streptomyces hygroscopicus.

5. Use of the engineered strain of Streptomyces hygroscopicus according to any one of claims 1 to 3 or the method for constructing the engineered strain of Streptomyces hygroscopicus according to claim 4 as or in the preparation of a drug for preventing and controlling biological diseases or a preservative for preventing and controlling spoilage; Preferably, the biological disease or spoilage is caused by bacterial, fungal pathogens or a combination thereof; More preferably, the biological disease or spoilage is caused by one or any combination of pathogenic fungi including Colletotrichum gloeosporioides, Fusarium oxysporum, and Phytophthora capsici.

6. Use of the engineered strain of Streptomyces hygroscopicus according to any one of claims 1 to 3 or the method for constructing the engineered strain of Streptomyces hygroscopicus according to claim 4 in the fermentative production of ε-polylysine.

7. The use according to claim 6, characterized in that Before fermentation to produce ε-polylysine, the engineered strain of Streptomyces hygroscopicus is subjected to adaptive evolution, wherein the engineered strain of Streptomyces hygroscopicus is subcultured every 24 to 48 hours in a TSB fermentation medium containing 0.5 to 3 g / L ε-polylysine for a total of 30 to 60 days.

8. A drug for preventing and treating biological diseases or a preservative for preventing and treating spoilage, characterized in that: Contains the engineered strain of Streptomyces hygroscopicus or its fermentation product according to any one of claims 1 to 3.

9. The drug for preventing and treating biological diseases or the preservative for preventing and treating spoilage according to claim 8, characterized in that: The fermented product is prepared by the following method: P1. After activating the engineered strain of Streptomyces hygroscopicus, inoculate it into a seed culture medium for cultivation to obtain a seed solution containing genetically recombinant bacteria. The seed solution OD 600 is 4.5-8.5; preferably, the seed culture medium is M3G or TSB medium; P2. The seed solution obtained in step P1 was inoculated into the fermentation medium at an inoculum size of 2% to 12% for fermentation in a fermenter; Preferably, the fermentation tank is fermented at a temperature of 28-37° C. and a shaking speed of 200 rpm for 48-96 hours to obtain a fermented product.

10. The biological disease control drug according to claim 9, characterized in that: The fermentation medium formula is as follows: Glucose 30-80g / L, soybean powder 5-15g / L, (NH4)2SO4 5-15g / L, yeast powder 2-8g / L, K2HPO4 0.5-1.0g / L, KH2PO4 0.5-1.5g / L, MgSO4·7H2O 0.4-0.8g / L, FeSO4·7H2O 0.02-0.04g / L, ZnSO4·7H2O 0.02-0.06g / L.

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