Method for improving spore production of natamycin producing strain

Abstract

The invention provides a method for improving spore production of natamycin producing bacteria, belongs to the technical field of streptomyces culture, and mainly provides an FtsZ gene, and the nucleotide sequence of the FtsZ gene is shown as SEQ ID NO.1; the FtsZ gene is applied to promotion of spore production of streptomyces gilvosporeus; the invention provides a genetically engineered bacterium for improving the sporulation quantity of streptomyces gilvosporeus. The genetically engineered bacterium is obtained by introducing an FtsZ gene and an erythromycin strong promoter ermEp into the streptomyces gilvosporeus through a carrier, a strain of streptomyces gilvosporeus F607-F is provided, the streptomyces gilvosporeus F607-F is preserved on October 28, 2024, and the preservation number of the streptomyces gilvosporeus F607-F is CCTCC M 20242352; the invention also provides a solid culture medium for promoting spore production of streptomyces gilvosporeus. The genetically engineered bacterium F607-F provided by the invention has the spore production advantage and can produce natamycin at high yield.

Description

Technical Field

[0001] The invention belongs to the technical field of streptomyces cultivation, and particularly relates to a method for improving spore production of natamycin producing bacteria. Background Art

[0002] Natamycin is an antifungal compound produced by the fermentation of Streptomyces. It belongs to the class of polyene macrolides and can widely inhibit the growth of molds and yeasts, as well as the production of mycotoxins. It is reported that in addition to its application in food and medicine, natamycin can also be used in animal husbandry, poultry farming and agriculture. In 1982, natamycin was officially approved as a food preservative by the US FDA. In 1997, my country officially approved natamycin as a food preservative. Currently, natamycin has been widely used in more than 50 countries.

[0003] Streptomyces chrysoporus is one of the industrial production strains of natamycin. It is an excellent industrial strain that, in addition to having excellent metabolic capacity, is also easy to expand and culture and has excellent storage stability. For Streptomyces chrysoporus, spores have better survival than mycelium and are easier to expand and culture for a long time and preserve. Therefore, obtaining a large number of spores is one of the prerequisites for the fermentation production stability of industrial production strains.

[0004] Streptomyces is a type of bacteria in the kingdom Bacteria and the order Actinomycetes. Most Streptomyces have hyphae and spores, and a few Streptomyces lose the ability to produce spores during multiple subcultures (Functions of Streptomyces and their application in agriculture, Zhu Zhiyan, etc.); when Streptomyces undergo strain mutation, they may also lose the ability to produce spores (Activation of recessive secondary metabolite biosynthesis gene clusters and targeted discovery of natural products, Hou Lukuan, Li Huayue, etc.). In industrial production, after Streptomyces loses the ability to produce spores, its reproductive capacity is limited. Spore formation is one of the important ways of reproduction of Streptomyces. Losing the ability to produce spores means that its reproductive efficiency is greatly reduced and the population grows slowly; the environmental adaptability decreases. The spores have extremely strong resistance to stress and can survive in harsh environments and wait for favorable conditions to germinate. Strains that do not produce spores may be more likely to die under drought, high temperature or other adverse conditions.

[0005] The study of genetic manipulation of Streptomyces began in the 1970s. Hopwood et al. successively used genetic transformation, phage transduction, transposon, electrotransformation and conjugation transfer to establish some genetic manipulation methods of Streptomyces. However, due to the great genetic differences among different Streptomyces, not all genetic manipulation methods are applicable. At present, there are three main methods for gene transfer in Streptomyces: transformation, conjugation transfer and phage transduction. However, due to the differences in transformation media, different growth conditions of hyphae and the existence of certain restriction modification systems in Streptomyces cells, the conjugation transfer method has become the preferred method for establishing the genetic manipulation system of Streptomyces.

[0006] Conjugation transfer is the process of exchanging plasmids between bacteria through direct or indirect contact. For Streptomyces, obtaining single-cell spores is one of the key factors for the success of the conjugation transfer method. Since the genetic engineering of Streptomyces chrysoporus is mostly achieved through conjugation transfer, and the conjugation transfer process requires a large number of spores, the increase in spore production has laid the foundation for the genetic engineering of Streptomyces chrysoporus. At the same time, the earlier spore production time also significantly reduces the genetic engineering time of Streptomyces chrysoporus.

[0007] Therefore, if Streptomyces loses its spore-forming ability, the application of the strain will be seriously affected. Restoring and improving the spore-forming ability of Streptomyces is of great significance.

[0008] The main uses of the FtsZ gene in the prior art are mainly concentrated in bacterial division and antibacterial drug research and development. FtsZ is a key protein in the process of bacterial cell division, and it plays a decisive role in bacterial division and proliferation. However, different FtsZ genes have certain differences in functional effects. For example, the Chinese document "Study on the Functional Characteristics of the Effector Molecule RxLR23 of Pepper Phytophthora and Its Key Developmental Regulatory Gene FtsZ" (Li Jing, Shandong Agricultural University, 2020) records two pepper Phytophthora orthologous genes, named PcFtsZ1 and PcFtsZ2. Through the heterologous expression of PcFtsZ1 and PcFtsZ2, this study found that the expression of PcFtsZ2 caused the bacterial cells to fail to divide normally and formed elongated filamentous cells, which is the same as the overexpression phenotype of EcFtsZ, but the expression of PcFtsZ1 has little effect on bacterial cell division.

[0009] Although actinomycetes belong to bacteria, there are significant differences in the reproduction methods of actinomycetes and bacteria. Bacteria mainly reproduce asexually, which is specifically manifested as transverse division of cells, called fission; actinomycetes mainly reproduce through asexual spores and hyphae fragments, mainly conidia; There are currently very few research reports on the FtsZ gene in actinomycetes, and there are no reports on the effect of FtsZ gene on the production of actinomycete spores. Summary of the invention

[0010] In view of the shortcomings of the prior art, the present invention provides a method for improving the spore production of natamycin producing bacteria.

[0011] A FtsZ gene, the nucleotide sequence of the FtsZ gene is shown as SEQ ID NO.1.

[0012] The above-mentioned FtsZ gene is used to promote the spore production of Streptomyces chrysoporus, and the nucleotide sequence of the FtsZ gene is shown in SEQ ID NO.1.

[0013] A genetically engineered bacterium for improving the spore production of Streptomyces chrysoporus, wherein the genetically engineered bacterium is obtained by introducing the FtsZ gene and the erythromycin strong promoter ermEp into the Streptomyces chrysoporus via a vector;

[0014] The nucleotide sequence of the FtsZ gene is shown in SEQ ID NO.1, and the nucleotide sequence of the erythromycin strong promoter ermEp is shown in SEQ ID NO.2.

[0015] A method for constructing a genetically engineered bacterium for increasing the spore production of Streptomyces chrysoporus comprises the following steps:

[0016] (1) Construction of recombinant plasmid pMS82-ermEp-F: The FtsZ gene and the erythromycin strong promoter ermEp were inserted into the multiple cloning site of the pMS82 vector plasmid to obtain the recombinant plasmid pMS82-ermEp-F;

[0017] The nucleotide sequence of the FtsZ gene is shown in SEQ ID NO.1, and the nucleotide sequence of the erythromycin strong promoter ermEp is shown in SEQ ID NO.2;

[0018] (2) Preparation of Escherichia coli ET12567 / pUZ8002 containing the recombinant plasmid pMS82-ermEp-F: The recombinant plasmid pMS82-ermEp-F obtained in step (1) was introduced into Escherichia coli DH5α, and the obtained transformants were sequenced for verification. Then, the vector plasmid with the correct sequencing result was introduced into Escherichia coli ET12567 / pUZ8002 to obtain Escherichia coli ET12567 / pUZ8002 containing the recombinant plasmid pMS82-ermEp-F;

[0019] (3) Preparation of genetically engineered bacteria for improving the spore production of Streptomyces chrysoporus: The Escherichia coli ET12567 / pUZ8002 containing the recombinant plasmid pMS82-ermEp-F obtained in step (2) is transferred into spores of Streptomyces chrysoporus by a conjugative transfer method to obtain genetically engineered bacteria for improving the spore production of Streptomyces chrysoporus.

[0020] A strain of Streptomyces sp. F607-F was deposited in the China Center for Type Culture Collection on October 28, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC M 20242352.

[0021] The use of the genetically engineered bacteria, the genetically engineered bacteria constructed by the above method or the Streptomyces chrysoporus F607-F in the production of natamycin.

[0022] A solid culture medium for improving the spore production of Streptomyces chrysoporus comprises soybean cake powder, glycerol, calcium carbonate, magnesium chloride, sodium phosphate and agar powder, and the solvent is water.

[0023] Preferably according to the present invention, the solid culture medium comprises 10-15 g / L soybean cake powder, 5-15 g / L glycerol, 0.25-1 g / L calcium carbonate, 0.25-1 g / L magnesium chloride, 0.5-2 g / L sodium phosphate and 15-20 g / L agar powder, and the solvent is water.

[0024] Further preferably, the solid culture medium comprises 12.5 g / L soybean cake powder, 10 g / L glycerol, 0.5 g / L calcium carbonate, 0.5 g / L magnesium chloride, 1 g / L sodium phosphate and 15-20 g / L agar powder, and the solvent is water.

[0025] The method for preparing the solid culture medium for improving the spore production of Streptomyces chrysoporus comprises the following steps:

[0026] First, grind the soybean cake powder, add water and boil it, then mix it with other components of the culture medium, adjust the pH to 7.2-7.4, and make up the volume.

[0027] According to the preferred embodiment of the present invention, in the above-mentioned preparation method, the prepared culture medium is sterilized at 115° C.-121° C. for 15-30 min, poured into a sterile bacterial culture vessel, and set aside after the solid culture medium solidifies.

[0028] The method for using the solid culture medium to culture Streptomyces chrysoporus comprises the following steps: inoculating Streptomyces chrysoporus onto the solid culture medium, and culturing until the Streptomyces chrysoporus produces spores.

[0029] Preferably according to the present invention, in the above method, Streptomyces chrysoporus is inoculated onto the solid culture medium by streaking.

[0030] The culture method of the genetically engineered bacteria, the genetically engineered bacteria constructed by the above method or the Streptomyces chrysoporus F607-F comprises the following steps: inoculating the genetically engineered bacteria, the genetically engineered bacteria constructed by the above method or the Streptomyces chrysoporus F607-F onto the solid culture medium and culturing until spores are produced.

[0031] Preferably according to the present invention, in the above-mentioned culture method, the above-mentioned genetically engineered bacteria, the genetically engineered bacteria constructed by the above-mentioned method or the above-mentioned Streptomyces chrysoporus F607-F is inoculated onto the above-mentioned solid culture medium by streaking.

[0032] The beneficial effects of the present invention include at least the following:

[0033] 1. The present invention provides for the first time an FtsZ gene that promotes spore production in Streptomyces chrysoporus, and the nucleotide sequence of the FtsZ gene is shown in SEQ ID NO.1.

[0034] 2. The present invention provides a genetically engineered strain of Streptomyces chrysoporus F607-F, which has a spore production advantage and can produce high levels of natamycin.

[0035] 3. The present invention provides a solid culture medium that is conducive to the spore production of Streptomyces chrysoporus. The solid culture medium provided by the present invention has a higher spore production than the Gaoshi No. 1 solid culture medium, the YMS solid culture medium, and the MS solid culture medium, and the culture time required for spore formation is also shorter. Since the genetic engineering of Streptomyces chrysoporus is mostly achieved through conjugation transfer, the unicellular spores are one of the key factors for the success of the conjugation transfer method, and the increase in spore production has laid a foundation for the genetic engineering of Streptomyces chrysoporus. At the same time, the earlier spore production time also significantly reduces the genetic engineering time of Streptomyces chrysoporus. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Plate images of mutant Streptomyces chrysoporus grown on different solid media.

[0037] Figure 2 Plate map of the mutant strain of Streptomyces chrysoporus corresponding to the optimization of carbon and nitrogen sources for solid culture medium.

[0038] Figure 3 Plate images of the original strain of Streptomyces chrysoporus grown on different solid media.

[0039] Figure 4 This is a plate map of the genetically engineered strain F607-F of Streptomyces chrysoporus cultured on MS solid medium. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0041] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0042] Unless otherwise specified, the experimental materials used in the following examples are all conventional biochemical reagents.

[0043] Vector pMS82 was purchased from Addgene;

[0044] Escherichia coli ET12567 / pUZ8002 was purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.;

[0045] Soybean cake powder was purchased from Beijing Hongrun Baoshun Technology Co., Ltd.

[0046] Streptomyces sp. F607 was used as the original bacterium for ARTP mutagenesis. A mutant strain was screened and its natamycin yield was increased by 25% compared with the original bacterium. However, due to the randomness of ARTP mutagenesis, other genes were changed, resulting in the mutant strain producing almost no spores on the original culture medium.

[0047] In order to continuously improve the production of natamycin by genetically engineering Streptomyces chrysoporus, the inventors sequenced the whole genome and transcriptome of the original bacteria and the mutant bacteria, and found that the transcriptional regulation of some genes was downregulated. Through blast, it was found that their gene sequences were similar to the FtsZ gene. The inventors designed and synthesized the FtsZ gene, and the nucleotide sequence of the FtsZ gene is shown in SEQ ID NO.1.

[0048] Because the mutant bacteria screened above hardly produce spores on the original culture medium, the inventors conducted research on a solid culture medium to increase the spore production of Streptomyces chrysoporus.

[0049] Example 1

[0050] A solid culture medium for increasing the spore production of Streptomyces chrysoporus is used for culturing Streptomyces chrysoporus, which is specifically as follows:

[0051] Prepare 1L of spore-forming solid culture medium: first grind the soybean cake powder, add tap water to boil, then mix with other components of the culture medium, adjust the pH to 7.2-7.4, fix the volume, and sterilize at 121℃ for 15min. Inoculate Streptomyces chrysogenum into the seed culture medium, the glucose content in the seed culture medium is 1% by mass, the peptone content is 0.6% by mass, the pH is 7.1, the solvent is water, and culture at 29℃ and 220rpm for 16-24h until the OD 600 =1.5.

[0052] Spore-producing solid culture medium: soybean cake powder 12.5g / L, glycerol 10g / L, calcium carbonate 0.5g / L, magnesium chloride 0.5g / L, sodium phosphate 1g / L, agar powder 20g / L, the solvent is water.

[0053] In the clean bench, pour the sterilized spore-producing solid culture medium into a sterile culture dish. After the solid culture medium solidifies, use a 1 ul sterile inoculation loop to take the Streptomyces chrysogenum seed liquid and streak it 10 times on the solid culture medium. Place the streaked Streptomyces chrysogenum culture dish in a 29°C constant temperature incubator for culture.

[0054] Example 2

[0055] Sporulation of the above-mentioned Streptomyces chrysoporus mutants in different culture media

[0056] Spore-forming solid culture medium: soybean cake powder 12.5g / L, glycerol 10g / L, calcium carbonate 0.5g / L, magnesium chloride 0.5g / L, sodium phosphate 1g / L, agar powder 20g / L, the solvent is water;

[0057] Gao's solid medium No. 1: purchased from Beijing Luqiao Technology Co., Ltd.;

[0058] YMS solid medium: yeast powder 4g / L, soluble starch 4g / L, maltose 10g / L, cobalt chloride 5mg / L, agar powder 20g / L, solvent is water;

[0059] MS solid culture medium: cooked soybean powder 20g / L, mannitol 20g / L, agar powder 20g / L, the solvent is water.

[0060] Prepare the above four solid culture media, adjust the pH to 7.2-7.4, fix the volume, and sterilize at 115℃ for 30min. Inoculate Streptomyces chrysogenum into the seed culture medium, culture at 29℃ and 220rpm for 16-24h, and culture until OD 600 =1.5. Use 1ul of sterile inoculation loop to pick up the seed liquid of Streptomyces chrysogenum and streak 10 times on the solid culture medium. Place the streaked culture dish in a 29℃ constant temperature incubator to observe the spore production of Streptomyces chrysogenum.

[0061] Spores of Streptomyces chrysogenus can be seen in 3 days of spore-forming solid medium. In Gao's No. 1 solid medium, YMS solid medium, and MS solid medium, only a very small number of spores are observed after 5-7 days of culturing. Figure 1 .

[0062] Figure 1 The medium-spore-producing solid culture medium plate is a plate that has been cultured for 3 days. It can be seen from the plate that the mutant bacteria of Streptomyces chrysoporus have produced more spores after being cultured for 3 days.

[0063] Figure 1 The Zhonggaoshi No. 1 solid culture medium plate is a plate cultured for 7 days, the YMS solid culture medium plate is a plate cultured for 7 days, and the MS solid culture medium plate is a plate cultured for 7 days. It can be seen from the plates that only a very small number of spores were observed.

[0064] Example 3

[0065] Optimization of carbon and nitrogen sources in solid culture medium for conidia production of Streptomyces chrysoporus

[0066] Production package solid culture medium: soybean cake powder 12.5g / L, glycerol 10g / L, calcium carbonate 0.5g / L, magnesium chloride 0.5g / L, sodium phosphate 1g / L, agar powder 20g / L, the solvent is water.

[0067] Culture medium 2: yeast powder 12.5g / L, glycerol 10g / L, calcium carbonate 0.5g / L, magnesium chloride 0.5g / L, sodium phosphate 1g / L, agar powder 20g / L, the solvent is water.

[0068] Culture medium 3: soybean cake powder 12.5g / L, soluble starch 10g / L, calcium carbonate 0.5g / L, magnesium chloride 0.5g / L, sodium phosphate 1g / L, agar powder 20g / L, the solvent is water.

[0069] Culture medium 4: yeast powder 12.5g / L, soluble starch 10g / L, calcium carbonate 0.5g / L, magnesium chloride 0.5g / L, sodium phosphate 1g / L, agar powder 20g / L, the solvent is water.

[0070] Prepare the above four solid culture media, adjust the pH to 7.2-7.4, fix the volume, and sterilize at 121℃ for 15min. Inoculate the above Streptomyces chrysogenus into the seed culture medium, culture at 29℃ and 220rpm for 16-24h, and culture until OD 600 =1.5, use 1ul of sterile inoculation loop to take the seed liquid of Streptomyces chrysogenum and streak 10 times on the solid culture medium. Place the streaked culture dish in a 29℃ constant temperature incubator to observe the spore production of Streptomyces chrysogenum.

[0071] Figure 2 It shows that more spores of Streptomyces chrysoporus can be seen in the spore-forming solid medium after 3 days of culture, while only a small number of spores were observed in other solid media after 7 days of culture.

[0072] Example 4

[0073] Optimization of the composition of solid culture medium for conidia production of Streptomyces chrysoporus

[0074] The invention provides a solid culture medium for Streptomyces chrysoporus that is conducive to spore production, comprising soybean cake powder, glycerol, calcium carbonate, magnesium chloride, sodium phosphate and agar powder.

[0075] Table 1

[0076]

[0077]

[0078] The solid culture medium (agar powder 20 g / L) prepared by method 1-11 was selected to determine the spore production, see Table 1.

[0079] The mutant strain of Streptomyces chrysogenum was inoculated into the seed culture medium and cultured at 29°C and 220 rpm for 16-24 h until OD 600=1.5, diluted 10 times, 100ul was spread on 11 different culture media, and the culture of the whole plate after 72-80h of culture was scraped and placed in a 15mL centrifuge tube with 10mL sterile water, fully shaken on a vortexer, filtered with double-layer gauze, mixed and diluted appropriately, and counted with a hemocytometer to calculate the spore production, see Table 2. Therefore, the content of each component in the spore-producing solid culture medium was ultimately the most suitable ratio based on method 1.

[0080] Table 2

[0081]

[0082] Example 5

[0083] Detection of spore activity in a solid culture medium for improving spore production of Streptomyces chrysoporus

[0084] The composition of the culture medium used was the same as that described in Example 2.

[0085] The spores growing well on the spore-producing solid medium, Gao's No. 1 solid medium, YMS solid medium, and MS solid medium were inoculated into the seed medium and cultured at 29°C and 220 rpm. The spores on the spore-producing solid medium and MS solid medium were cultured in the seed medium for 16 h, and the OD 600 =2.0; spores on Gao's No. 1 solid medium and YMS solid medium were cultured in seed medium for 16 h, OD 600 =1.4. It can be seen that the spore activity on the spore-producing solid medium is comparable to that on the MS solid medium, and is better than that on the Gaoshi No. 1 solid medium and the YMS solid medium.

[0086] Example 6

[0087] Effect of Solid Sporulation Medium on Sporulation of Primary Bacteria

[0088] The composition of the culture medium used was the same as that described in Example 2.

[0089] Inoculate Streptomyces chrysogenum F607 into the seed culture medium and culture at 29°C and 220 rpm for 16-24 h until OD 600 =1.5.

[0090] In the clean bench, pour the sterilized solid culture medium into a sterile culture dish. After the solid culture medium solidifies, use a 1ul sterile inoculation loop to pick up the seed liquid of Streptomyces chrysogenum and streak it 10 times. Place the streaked culture dish in a 29℃ constant temperature incubator to observe the spore production of Streptomyces chrysogenum. Figure 3 , is the plate image of 5 days of culture, Figure 3It can be seen that the spore-forming solid culture medium provided by the present invention is more conducive to the spore production of Streptomyces chrysoporus.

[0091] Example 7

[0092] Construction of a genetically engineered strain of Streptomyces chrysoporus with high spore production

[0093] 1. Construction of recombinant plasmid pMS82-ermEp-F

[0094] (1) Design and synthesize the FtsZ gene and the erythromycin strong promoter ermEp gene, amplify the FtsZ gene and the erythromycin strong promoter ermEp gene fragments, and recover and purify them for later use.

[0095] The nucleotide sequence of the FtsZ gene is shown in SEQ ID NO.1, and the nucleotide sequence of the erythromycin strong promoter ermEp is shown in SEQ ID NO.2.

[0096] (2) After analyzing the sequences of the vector pMS82 and the inserted fragment, the inserted fragment was connected to the vector by homologous recombination. In order to increase the concentration of the vector, the PCR amplification linearization method was selected to perform reverse PCR amplification on the vector plasmid. The linearized vector pMS82 was amplified and purified by gel recovery for later use.

[0097] (3) The insert fragment and the vector plasmid were connected using a homologous recombination kit. The molar ratio of the linearized vector plasmid pMS82 to the insert fragment was calculated according to the instructions. After pipetting and mixing, the mixture was reacted at 50° C. for 30 min to obtain the recombinant plasmid pMS82-ermEp-F.

[0098] 2. Preparation of E. coli ET12567 / pUZ8002 containing recombinant plasmid pMS82-ermEp-F

[0099] (1) Take a competent cell of E. coli DH5α and place it in the culture medium. Thaw it on ice, add 10ul of recombinant plasmid pMS82-ermEp-F, place it on ice for 30min, heat shock it in a 42℃ water bath for 90s, place it on ice for 2min, add 500ul of antibiotic-free LB liquid culture medium, and culture it at 37℃ and 180rpm for 1h. Spread it on a solid LB plate containing 200ng / ul hygromycin resistance, and culture it at 37℃ for 16h until a single colony of E. coli DH5α containing the recombinant plasmid pMS82-ermEp-F grows. Pick a single colony with good growth for expansion culture, perform bacterial liquid PCR verification, send it to a third-party sequencing company for sequencing and sequence comparison, and extract and verify the correct recombinant plasmid pMS82-ermEp-F.

[0100] (2) Take a competent cell of E. coli ET12567 / pUZ8002 and place it in the culture medium. Thaw it on ice, add 10ul of the verified recombinant plasmid pMS82-ermEp-F, place it on ice for 30min, heat shock it in a 42℃ water bath for 90s, place it on ice for 2min, add 500ul of antibiotic-free LB liquid culture medium, and culture it at 37℃ and 180rpm for 1h. Spread it on a solid LB plate containing 25ng / ul kanamycin, 25ng / ul chloramphenicol, and 200ng / ul hygromycin resistance, and culture it at 37℃ for 16h until a single colony of E. coli ET12567 / pUZ8002 containing the recombinant plasmid pMS82-ermEp-F grows. Pick a single colony with good growth for expansion culture and perform bacterial liquid PCR verification.

[0101] (3) Inoculate the E. coli ET12567 / pUZ8002 introduced with the recombinant plasmid pMS82-ermEp-F into 5 ml LB liquid medium containing 25 ng / ul kanamycin, 25 ng / ul chloramphenicol, and 200 ng / ul hygromycin, and culture at 37°C, 180 rpm for 16 h. Transfer to a fresh 20 ml LB liquid medium containing 25 ng / ul kanamycin, 25 ng / ul chloramphenicol, and 200 ng / ul hygromycin, and culture at 37°C, 180 rpm until OD600 = 0.4-0.6. Transfer to a sterile 50 ml centrifuge tube, centrifuge at 4000 rpm, 4°C for 5 min, and discard the supernatant. Wash twice with 10 ml of antibiotic-free LB liquid medium to wash off the antibiotics, and resuspend in 250 ul LB liquid medium for later use.

[0102] 3. Preparation of genetically engineered strains of Streptomyces chrysogenum

[0103] (1) The mutant strain of Streptomyces chrysogenum was inoculated into the seed culture medium and cultured at 29°C and 220 rpm until OD 600 =2.0. Use 1ul of sterile inoculation loop to take the seed liquid of Streptomyces chrysogenum and streak it 10 times on the spore-forming solid medium. Place the streaked Streptomyces chrysogenum culture dish at 29℃ for 3 days. Scrape the spores into 5ml of 0.05M, pH 8.0 TES solution. Heat shock the centrifuge tube in a 50℃ water bath for 10min, then cool it to room temperature with running tap water. Add 5ml of 2× spore pre-germination medium to the tube, then place the centrifuge tube in a 29℃ shaker and culture at 200rpm for 3h. After culture, centrifuge at 4000rpm for 10min, remove the supernatant, wash twice with 10ml of antibiotic-free LB liquid medium, then add 250ul of antibiotic-free LB medium to the tube to suspend the spores for later use.

[0104] (2) Mix 250ul of Escherichia coli ET12567 / PUZ8002 bacterial suspension and 250ul of spore suspension, spread on MS solid culture medium, blow dry in a clean bench until there is no water stain, and then invert the plate in a 29℃ incubator for 20h. Take 1ml of sterile water, add 1mg of nalidixic acid and hygromycin, mix, and then evenly cover the surface of the solid culture medium, blow dry in a clean bench until there is no water stain, invert in a 29℃ incubator for culturing, and single conjugated colonies can be seen in 3-4 days. Transfer the single colony to a new MS solid culture medium for purification and culture for two rounds, select the single colony that produces white spores to extract genomic DNA, and use the genomic DNA as a template for PCR verification to obtain the genetically engineered strain F607-F of Streptomyces chrysogenum with high spore production. Figure 4 In combination with the plate transferred to MS solid medium and cultured for 7 days, single colonies producing white spores were observed.

[0105] The above-mentioned Streptomyces chrysogenus mutant was cultured on MS solid medium plates for 7 days (see Figure 1 ), only a small amount of spores were observed, and the genetically engineered strain F607-F of Streptomyces chrysogenum was cultured on MS solid medium plates for 7 days (see Figure 4 ), it can be observed that more spores have been produced; the genetically engineered strain F607-F of Streptomyces chrysoporus has obvious spore production advantages.

[0106] The genetically engineered strain F607-F of Streptomyces chrysoporus obtained above was preserved. The specific preservation information is as follows:

[0107] A strain of Streptomyces sp. F607-F was deposited in the China Center for Type Culture Collection on October 28, 2024, with the deposit number: CCTCC M 20242352, and the deposit address is Wuhan University, Wuhan, China.

[0108] Example 8

[0109] Application of genetically engineered Streptomyces chrysogenum F607-F and mutant strain of Streptomyces chrysogenum in producing natamycin

[0110] (1) Seed liquid culture: The genetically engineered strain F607-F of Streptomyces chrysogenum and the mutant strain of Streptomyces chrysogenum were inoculated into the seed culture medium and cultured at 29°C and 220 rpm for 16-24 h until the OD 600 =1.5-2.0, obtain seed solution.

[0111] (2) Fermentation culture: The genetically engineered Streptomyces fuchsporus F607-F and the mutant Streptomyces fuchsporus seed solution in step (1) are inoculated into the fermentation medium at an inoculum concentration of 2% by volume, and the fermentation culture is carried out at a temperature of 29° C. and a rotation speed of 220 rpm for 110-120 hours to obtain a fermentation liquid containing natamycin, and the fermentation liquid is separated and purified to obtain natamycin. The fermentation medium contains 7% glucose by mass, 2% peptone by mass, water as the solvent, a pH of 7.1, and sterilization at 115° C. for 30 minutes. The obtained natamycin yield is shown in Table 3, and experimental groups 1, 2, and 3 are repeated three times. The genetically engineered Streptomyces fuchsporus F607-F provided by the present invention has a spore production advantage and can produce high natamycin.

[0112] Table 3

[0113] Natamycin yield (g / L) Streptomyces chrysogenum genetic engineering bacteria experimental group 1 14.8 Streptomyces chrysogenum genetic engineering bacteria experimental group 2 15.2 Streptomyces chrysogenum genetic engineering bacteria experimental group 3 15.5 Streptomyces chrysogenum mutagenesis group 1 14.5 Streptomyces chrysogenum mutagenesis group 2 15.3 Streptomyces chrysogenum mutagenesis group 3 15.6

[0114] The present invention provides for the first time an FtsZ gene that promotes spore production of Streptomyces chrysoporus, and the nucleotide sequence of the FtsZ gene is shown in SEQ ID NO. 1. The present invention provides a genetically engineered strain of Streptomyces chrysoporus F607-F, which has a spore production advantage and can produce high levels of natamycin.

[0115] The present invention also provides a solid culture medium that is beneficial to the spore production of Streptomyces chrysoporus. The solid culture medium provided by the present invention is higher than the Gaoshi No. 1 solid culture medium, the YMS solid culture medium, and the MS solid culture medium. The spore production of Streptomyces chrysoporus is greater, and the culture time required for spore formation is also shorter.

Claims

1. A FtsZ gene, characterized in that: The nucleotide sequence of the FtsZ gene is shown in SEQ ID NO.

1.

2. Use of the FtsZ gene according to claim 1 in promoting conidia production in Streptomyces chrysoporus.

3. A genetically engineered bacterium for increasing the spore production of Streptomyces chrysoporus, characterized in that: The genetically engineered bacteria are obtained by introducing the FtsZ gene and the erythromycin strong promoter ermEp into Streptomyces chrysosporium through a vector; The nucleotide sequence of the FtsZ gene is shown in SEQ ID NO.1, and the nucleotide sequence of the erythromycin strong promoter ermEp is shown in SEQ ID NO.

2.

4. The method for constructing a genetically engineered bacterium for increasing the spore production of Streptomyces chrysoporus according to claim 3, characterized in that: The steps include: (1) Construction of recombinant plasmid pMS82-ermEp-F: The FtsZ gene and the erythromycin strong promoter ermEp were inserted into the multiple cloning site of the pMS82 vector plasmid to obtain the recombinant plasmid pMS82-ermEp-F; The nucleotide sequence of the FtsZ gene is shown in SEQ ID NO.1, and the nucleotide sequence of the erythromycin strong promoter ermEp is shown in SEQ ID NO.2; (2) Preparation of Escherichia coli ET12567 / pUZ8002 containing the recombinant plasmid pMS82-ermEp-F: The recombinant plasmid pMS82-ermEp-F obtained in step (1) was introduced into Escherichia coli DH5α, and the obtained transformants were sequenced for verification. Then, the vector plasmid with the correct sequencing result was introduced into Escherichia coli ET12567 / pUZ8002 to obtain Escherichia coli ET12567 / pUZ8002 containing the recombinant plasmid pMS82-ermEp-F; (3) Preparation of genetically engineered bacteria for improving the spore production of Streptomyces chrysoporus: The Escherichia coli ET12567 / pUZ8002 containing the recombinant plasmid pMS82-ermEp-F obtained in step (2) is transferred into spores of Streptomyces chrysoporus by a conjugative transfer method to obtain genetically engineered bacteria for improving the spore production of Streptomyces chrysoporus.

5. A strain of Streptomyces sp. F607-F was deposited in the China Center for Type Culture Collection on October 28, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC M 20242352.

6. Use of the genetically engineered bacteria according to claim 3, the genetically engineered bacteria constructed by the method according to claim 4, or the Streptomyces chrysoporus F607-F according to claim 5 in the production of natamycin.

7. A solid culture medium for increasing the spore production of Streptomyces chrysoporus, characterized in that: The ingredients are soybean cake powder, glycerol, calcium carbonate, magnesium chloride, sodium phosphate and agar powder, and the solvent is water.

8. The solid culture medium according to claim 7, characterized in that The ingredients are 10-15 g / L soybean cake powder, 5-15 g / L glycerol, 0.25-1 g / L calcium carbonate, 0.25-1 g / L magnesium chloride, 0.5-2 g / L sodium phosphate and 15-20 g / L agar powder, and the solvent is water; Preferably, the ingredients are 12.5 g / L soybean cake powder, 10 g / L glycerol, 0.5 g / L calcium carbonate, 0.5 g / L magnesium chloride, 1 g / L sodium phosphate and 15-20 g / L agar powder, and the solvent is water; Preferably, the method for preparing the solid culture medium comprises the following steps: First, grind the soybean cake powder, add water and boil it, then mix it with other components of the culture medium, adjust the pH to 7.2-7.4, and make up the volume; Preferably, in the preparation method, the prepared culture medium is sterilized at 115° C.-121° C. for 15-30 min, poured into a sterile bacterial culture vessel, and set aside after the solid culture medium solidifies.

9. The method for culturing Streptomyces chrysoporus using the solid culture medium according to any one of claims 7 to 8, characterized in that: The method comprises the following steps: inoculating Streptomyces chrysogenum onto a solid culture medium, and culturing until the Streptomyces chrysogenum produces spores; Preferably, Streptomyces chrysoporus is inoculated onto a solid culture medium by streaking.

10. The method for culturing the genetically engineered bacteria according to claim 3, the genetically engineered bacteria constructed by the method according to claim 4, or the Streptomyces chrysoporus F607-F according to claim 5, characterized in that: The method comprises the following steps: inoculating the genetically engineered bacteria according to claim 3, the genetically engineered bacteria constructed by the method according to claim 4, or the Streptomyces chrysoporus F607-F according to claim 5 onto the solid culture medium according to any one of claims 7 to 8, and culturing until spores are produced; Preferably, the cells are inoculated onto a solid culture medium by streaking.

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