Recombinant streptomyces gilvosporeus for producing high-purity natamycin as well as construction method and application of recombinant streptomyces gilvosporeus
By introducing specific genes into Streptococcus brilliformis and constructing recombinant strains, the problem of intermediate volume accumulation in fermentation production is solved, and efficient production of high-purity natamycin is achieved, which reduces production costs.
Patent Information
- Application Number
- CN202510109525.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-23
- Publication Date
- 2025-04-04
AI Technical Summary
When the prior art produces natamycin by fermenting and producing natamycin, the accumulation of intermediate volume leads to increased energy consumption and difficulty in purification, and the yield is low, resulting in a higher price of natamycin, limiting its market application.
By introducing the encoding genes of glycosyltransferase, P450 monooxygenase and efflux pump protein into Streptocytica thorax, a recombinant strain was constructed to achieve complete transformation of the intermediate and efficient transport of natamycin.
The complete conversion of intermediates is achieved, the metabolic map of the fermentation broth is simplified, the purity and yield of natamycin are improved, and the production cost is reduced. It is suitable for large-scale industrial fermentation production.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of genetic engineering, and specifically relates to a recombinant Streptomyces chrysoporus for producing high-purity natamycin, and a construction method and application thereof. Background Art
[0002] Natamycins are well-known for their good broad-spectrum antifungal effects. They have been used as preservatives in the food industry for more than 30 years. They are a class of natural polyene macrolide antibiotics isolated from Streptomyces. Natamycins bind to ergosterol on the fungal cell membrane, changing the permeability of the cell membrane, allowing small molecules and ions inside the cell to escape, thereby causing fungal cell death. In addition to being used in the food industry, natamycins can also be used in the medical and health field to treat human fungal infections, and in the agricultural and animal husbandry fields as biological pesticides and silage additives to prevent and control fungi.
[0003] The biosynthesis of natamycin starts with acetyl-CoA, and the extension of the polyketide chain is catalyzed by PKS I polyketide synthase, including 11 malonyl-CoA and 1 methylmalonyl-CoA extension units, followed by the formation of a six-membered small ring and a hexadecene amide macrocycle by thioesterase, which are then released, and finally undergo carboxylation catalyzed by cytochrome P450 enzymes, glycosylation catalyzed by glycosyltransferases, and post-epoxidation modification reactions catalyzed by P450 monooxygenases to form natamycin. The sugar group is trehaloseamine, which is formed by dehydration, isomerization, and transamination of GDP-mannose.
[0004] In the process of using artificial food preservatives, people gradually found that they have a great impact on human health. In recent years, the advantages of natural preservatives have been gradually discovered and gradually become a hot topic of research. Therefore, the demand for natamycin in the domestic and foreign markets has been growing continuously. However, due to the high energy consumption of conventional fermentation and the relatively low yield, its price is relatively high compared with artificial foods such as potassium sorbate and sodium benzoate. It is currently mainly used in high value-added foods. How to reduce costs and promote the use of natamycin and further expand the market is a current problem.
[0005] In actual industrial production, in addition to the main product natamycin, the accumulation of intermediates is caused by insufficient supply of key enzymes in the synthesis process ( Figure 1 ), the intermediates often lose their antifungal activity due to the absence of key antibacterial groups (carboxyl at C12, trehalose amino group at C15, and epoxy group at C3-C5), and their accumulation increases the energy consumption and difficulty of subsequent separation and purification.
[0006] It is of great significance to study and develop a strain that has no intermediate accumulation in the fermentation broth during the fermentation production of natamycin and has a certain advantage in natamycin production. Summary of the invention
[0007] In view of the deficiencies in the prior art, the present invention provides a recombinant Streptomyces chrysoporus for producing high-purity natamycin, and a construction method and application thereof.
[0008] The purpose of the present invention is to provide a recombinant Streptomyces chrysoporus strain capable of fully converting intermediates, producing high-purity natamycin, and efficiently transporting natamycin to the extracellular space, as well as a construction method and application thereof.
[0009] The technical solution of the present invention is as follows:
[0010] A recombinant Streptomyces chrysoporus for producing high-purity natamycin, wherein the recombinant Streptomyces chrysoporus is obtained by introducing a glycosyltransferase encoding gene sgnk, a P450 monooxygenase encoding gene sgnd and an efflux pump protein encoding gene sgnh into the Streptomyces chrysoporus in combination with a promoter through a vector;
[0011] The nucleotide sequence of the gene sgnk is shown as SEQ ID NO.1, the nucleotide sequence of the gene sgnd is shown as SEQ ID NO.2, and the nucleotide sequence of the gene sgnh is shown as SEQ ID NO.3.
[0012] Preferably according to the present invention, the nucleotide sequence of the promoter is shown as SEQ ID NO.4.
[0013] The method for constructing the recombinant Streptomyces chrysoporus for producing high-purity natamycin comprises any of the following schemes:
[0014] Scheme 1: The method for constructing the recombinant Streptomyces chrysoporus comprises the following steps:
[0015] (1) Using the genomic DNA of Streptomyces chrysogenum as a template, PCR amplification was performed to obtain the sgnk gene fragment, and using the pSET152 plasmid as a template, PCR amplification was performed to obtain the Pint promoter fragment. After the two fragments were purified, they were spliced with the pMS82 linear vector. The spliced products were transformed into Escherichia coli. After verification, the obtained transformants were extracted to obtain the pMS82-Pint-sgnk plasmid;
[0016] The nucleotide sequence of the Pint promoter fragment is shown in SEQ ID NO.4;
[0017] (2) Obtain pMS82-Pint-sgnd and pMS82-Pint-sgnh plasmids using the same method as step (1);
[0018] (3) The pMS82-Pint-sgnk plasmid was digested with MfeI and SpeI to obtain the Pint-sgnk-Mfe / Spe fragment, and the pMS82-Pint-sgnd plasmid was digested with XbaI and PstI to obtain the Pint-sgnd-Xba / Pst fragment. The obtained fragments Pint-sgnk-Mfe / Spe and Pint-sgnd-Xba / Pst were enzymatically linked with pMS82-Mfe / Pst, and the ligation system was transformed into Escherichia coli. After verification, the obtained transformants were extracted to obtain the pMS82-Pint-sgnk-Pint-sgnd plasmid;
[0019] (4) The pMS82-Pint-sgnk-Pint-sgnd plasmid was digested with MfeI and SpeI to obtain the Pint-sgnk-Pint-sgnd-Mfe / Spe fragment, and the pMS82-Pint-sgnh plasmid was digested with XbaI and PstI to obtain the Pint-sgnh-Xba / Pst fragment. The two fragments were ligated with the pMS82-Mfe / Pst enzyme, and the ligation system was transformed into Escherichia coli. After verification, the obtained transformants were extracted to obtain the pMS82-Pint-sgnk-Pint-sgnd-Pint-sgnh plasmid;
[0020] (5) Transform the plasmid obtained in step (4) into the competent medium of ET12567 / pUZ8002 to obtain the donor bacteria ET12567 / pUZ8002 / pMS82-Pint-sgnk-Pint-sgnd-Pint-sgnh;
[0021] (6) performing conjugation transfer between the donor bacteria obtained in step (5) and Streptomyces chrysoporus to obtain a conjugate, and after purification and PCR verification of the conjugate, obtaining a recombinant Streptomyces chrysoporus;
[0022] Scheme 2: The method for constructing the recombinant Streptomyces chrysoporus comprises the following steps:
[0023] ① The gene sgnk, gene sgnd, gene sgnh and Pint promoter were biosynthesized to obtain the Pint-sgnk-Pint-sgnd-Pint-sgnh long fragment, which was spliced with the pMS82 linear vector by seamless cloning, and the splicing system was transformed into Escherichia coli. After verification, the obtained transformants were extracted to obtain the pMS82-Pint-sgnk-Pint-sgnd-Pint-sgnh plasmid;
[0024] The nucleotide sequence of the Pint promoter fragment is shown in SEQ ID NO.4;
[0025] ②Transform the plasmid obtained in step ① into the competent medium of ET12567 / pUZ8002 to obtain the donor bacteria ET12567 / pUZ8002 / pMS82-Pint-sgnk-Pint-sgnd-Pint-sgnh;
[0026] ③ The donor bacteria obtained in step ② are conjugated with Streptomyces chrysoporus to obtain conjugates, and the conjugates are purified and PCR verified to obtain recombinant Streptomyces chrysoporus.
[0027] A Streptomyces sp. NTIII-KDH, deposited on October 28, 2024, deposited by China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20242353, and a deposit address of Wuhan University, Wuhan, China.
[0028] Use of the above recombinant Streptomyces chrysoporus, the recombinant Streptomyces chrysoporus constructed by the above method or the above Streptomyces chrysoporus NTIII-KDH in the preparation of natamycin.
[0029] The method for preparing natamycin by the above-mentioned recombinant Streptomyces chrysoporus, the recombinant Streptomyces chrysoporus constructed by the above-mentioned method or the above-mentioned Streptomyces chrysoporus NTIII-KDH comprises the following steps:
[0030] The Streptomyces chrysoporus is activated, then inoculated into a seed culture medium and cultured to a logarithmic phase, inoculated into a fermentation medium at a volume ratio of 1-1.5%, and fermented at 28-30° C. for 4-5 days to obtain a fermentation liquid, which is purified to obtain natamycin.
[0031] Preferably according to the present invention, the components of the seed culture medium include: yeast powder 2-4 g / L, peptone 4-6 g / L, glucose 4-6 g / L, NaCl 2-6 g / L, pH 7.0-7.4, and the solvent is water.
[0032] Preferably according to the present invention, the components of the fermentation medium include: 4-7 g / L yeast powder, 15-25 g / L peptone, 15-20 g / L glucose, 1-3 g / L NaCl, and the solvent is water.
[0033] The beneficial effects of the present invention include at least the following:
[0034] 1. The recombinant Streptomyces NTIII-KDH provided by the present invention realizes the complete conversion of intermediates and the simplification of the metabolic profile of the fermentation broth during the fermentation process, while improving the active transport capacity of natamycin to the extracellular space and alleviating the toxic pressure. The bacterium has a better growth state during the entire fermentation process and is suitable for industrial large-scale fermentation production.
[0035] 2. The purity of natamycin prepared by fermentation of the recombinant Streptomyces chrysoporus NTIII-KDH provided by the present invention is as high as over 99%. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 This is a structural diagram of the intermediate product of the NTIII strain in the present invention.
[0037] Figure 2 The figure is a schematic diagram of the construction process of the plasmid for co-expression of sgnk gene, sgnd gene and sgnh gene in the present invention.
[0038] Figure 3 This is an agarose gel electrophoresis diagram of PCR verification of the NTIII-KDH engineering bacteria in the present invention.
[0039] Figure 4 This is a HPLC detection chart of the fermentation stock solution of the NTIII-KDH engineered bacteria and the control bacteria NTIII provided by the present invention.
[0040] Figure 5 This is an HPLC detection diagram of the product obtained by purifying the fermentation broth of the NTIII-KDH engineered bacteria and the control bacteria NTIII provided by the present invention. DETAILED DESCRIPTION
[0041] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.
[0042] The experimental methods without specific conditions and reagents without formulas in the following examples are all conventional conditions in the art.
[0043] The Streptomyces gilvosporeus NTIII used below is a mutant strain obtained by multiple rounds of ARTP (normal temperature and pressure plasma) mutagenesis screening of Streptomyces gilvosporeus F607. This strain has superior performance and certain advantages in natamycin production.
[0044] Example 1. Construction of plasmids pMS82-Pint-sgnk, pMS82-Pint-sgnd, and pMS82-Pint-sgnH
[0045] The genomic DNA of Streptomyces NTIII was used as a template to perform high-fidelity PCR amplification to obtain the sgnk (SEQ ID NO.1), sgnd (SEQ ID NO.2) and sgnh (SEQ ID NO.3) gene fragments, and the pSET152 (a conventional universal integration vector for Streptomyces, purchased from Miaoling Bio Wuhan Miaoling Biotechnology Co., Ltd.) plasmid was used as a template to perform PCR amplification to obtain the Pint promoter fragment (SEQ ID NO.4). The primer sequences are as follows:
[0046] Primer sequences for amplifying sgnk:
[0047] KF:5'-CACAAGGGGTTGTGACCGGGATGGAATCCGCCCGACGGCC-3'SEQ ID NO.5;
[0048] KR:5'-GACTGCAGATGCATACTAGTCGCCAAGGGCCGGGAGCGAGA-3'SEQ ID NO.6;
[0049] Primer sequences for amplifying sgnd:
[0050] DF:5'-CACAAGGGGTTGTGACCGGGACCGCCGCCTCCCACGACCT-3'SEQ ID NO.7;
[0051] DR:5'-GACTGCAGATGCATACTAGTCTACCAGACCACCCGGAGCT-3'SEQ ID NO.8;
[0052] Primer sequences for amplifying sgnh:
[0053] HF:5'-CACAAGGGGTTGTGACCGGGATGAGCCTCTGCCGCCGCGT-3'SEQ ID NO.9;
[0054] HR:5'-GACTGCAGATGCATACTAGTTCAGGAAGTCGACTGATGGG-3'SEQ ID NO.10;
[0055] Primer sequences for amplifying Pint promoter fragment:
[0056] Pint-F:5'-TCGAATTCCAATTGTCTAGAGCCGGAGCAATCGCCCTGGG-3'SEQ ID NO.11;
[0057] Pint-R:5'-CCCGGTCACAACCCCTTGTG-3'SEQ ID NO.12.
[0058] The PCR amplification system was: DNA template 10-50 ng, primer 10 μM 2 μL, 2× phanta Max Master Mi x25 μL, water 20 μL;
[0059] PCR amplification conditions were as follows: 1) 95°C, 3 min, pre-denaturation; 2) 95°C, 3 min, denaturation; 3) 60°C, 15 sec, annealing; 4) 72°C, 1-2 min; steps 2-4 were repeated for 35 cycles, and finally extended at 72°C for 5 min.
[0060] The obtained PCR products were recovered and purified by a conventional gel recovery kit to obtain sgnk, sgnd, sgnh and Pint fragments. The sgn k, sgnd and sgnh fragments were respectively spliced with the Pint fragment and the pMS82 (a conventional universal integrative vector for Streptomyces, purchased from addgene) linear vector using the Norwegian seamless cloning kit. The splicing products were chemically transformed into DH5α Escherichia coli competent cells, respectively, and spread on LB (200 ng / mL hygromycin) solid culture, and cultured at 37°C. After verification, the obtained transformants were extracted to obtain pMS82-Pint-sgnk, pMS82-Pint-sgnd and pMS82-Pint-sgnh plasmids.
[0061] Example 2. Construction of plasmid pMS82-Pint-sgnk-Pint-sgnd-Pint-sgnh
[0062] The plasmid pMS82-Pint-sgnk was digested with MfeI and SpeI to obtain the Pint-sgnk-Mfe / Spe fragment, and the pMS82-Pint-sgnd plasmid was digested with XbaI and PstI to obtain the Pint-sgnd-Xba / Pst fragment; Pint-sgnk-Mfe / Spe, Pint-sgnd-Xba / Pst and pMS82-Mfe / Pst were enzymatically linked, and the ligation system was chemically transformed into DH5α Escherichia coli competent cells, spread on LB (200 ng / mL hygromycin) solid culture, and cultured at 37°C. After verification, the obtained transformants were extracted to obtain the pMS82-Pint-sgn k-Pint-sgnd plasmid.
[0063] The plasmid pMS82-Pint-sgnk-Pint-sgnd was digested with MfeI and SpeI to obtain Pint-sgnk-Pint-sgnd-Mfe / Spe fragment, and the pMS82-Pint-sgnh plasmid was digested with XbaI and PstI to obtain Pint-sgnh-Xba / Pst fragment. The two fragments were ligated with pMS82-Mfe / Pst enzyme, and the ligation system was chemically transformed into DH5α Escherichia coli competent cells, spread on LB (200 ng / mL hygromycin) solid culture medium, and cultured at 37°C. After verification, the obtained transformants were extracted to obtain the pMS82-Pint-sgnk-Pint-sgnd-Pi nt-sgnh plasmid ( Figure 2 ).
[0064] Example 3: Construction of engineered bacteria NTIII-KDH
[0065] The expression plasmid pMS82-Pint-sgnk-Pint-sgnd-Pint-sgnh was chemically transformed into the competent state of Escherichia coli ET12567 / pUZ8002 (conventional general tool strain, purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.) to obtain the ET12567 / pU Z8002 / pMS82-Pint-sgnk-Pint-sgnd-Pint-sgnh donor bacteria, and the donor bacteria were transferred by conjugation with Streptomyces chrysosporium NTIII, and spread on MS (10mM MgCI2) plates, inverted cultured at 28°C for 18-20h, and 1mg nalidixic acid and 1mg hygromycin were covered on the plates. After inverted culture at 28°C for 3-5d, white conjugates were visible. The conjugates were transferred to MS solid culture medium for purification and culture for two rounds, and the genomic DNA of the conjugates was extracted as a template, and PCR verification was performed using ver-F / R verification primers. The PCR product electrophoresis detection was consistent with the expected size of 4721bp ( Figure 3 ), and the engineered bacteria NTIII-KDH was obtained.
[0066] The MS solid culture medium comprises: 20 g / L soybean cake powder, 20 g / L mannitol, pH 7.0-7.4, 1.5-2% agar, the solvent is water, and is sterilized by high pressure at 121° C. for 20 min.
[0067] The verification primers are as follows:
[0068] ver-F:5'-CCGGCGGACGGTTCGGCCCC-3'SEQ ID NO.13;
[0069] ver-R:5'-GTATGTTGTGTGGAATTGTG-3'SEQ ID NO.14;
[0070] PCR amplification conditions were as follows: 1) 95°C, 3 min, pre-denaturation; 2) 95°C, 3 min, denaturation; 3) 60°C, 15 sec, annealing; 4) 72°C, 30 sec, extension; steps 2-4 were repeated for 35 cycles, and finally extension was performed at 72°C for 5 min.
[0071] The engineered bacteria NTIII-KDH was deposited, and the specific deposit information is as follows:
[0072] A Streptomyces sp. NTIII-KDH, deposited on October 28, 2024, deposited by China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20242353, and a deposit address of Wuhan University, Wuhan, China.
[0073] Example 4: Fermentation culture of engineered bacteria NTIII-KDH
[0074] (1) Seed culture: Take the glycerol bacteria frozen at -80℃ and inoculate them into the seed solid plate for activation culture at 28℃ for 3-5 days. The fresh mycelium grown is transferred to the seed medium and cultured with shaking until the logarithmic growth phase. Seed medium: 3g / L yeast powder, 5g / L peptone, 5g / L glucose, 3g / L NaCl, pH 7.0-7.4, the solvent is water, and 1.5%-2% agar powder should be added to the seed solid plate.
[0075] (2) Fermentation culture: The seed liquid grown to the logarithmic growth phase is inoculated into the fermentation medium at a volume ratio of 1%, and the fermentation culture is carried out at 28-30°C and 220 rpm for 110-120 hours. The fermentation liquid is further purified to obtain natamycin. The fermentation medium: 5 g / L yeast powder, 20 g / L peptone, 20 g / L glucose, 2 g / L NaCl, and the solvent is water.
[0076] (3) Detection of Natamycin
[0077] Detection of fermentation stock solution: Take 1 mL of fermentation stock solution, add 60% methanol to make up to 10 mL, ultrasonicate for 30 min, take 1 mL and transfer to a 1.5 mL centrifuge tube and centrifuge at 12000 rpm for 10 min. The obtained supernatant is filtered through a 0.45 μm membrane filter and detected by high performance liquid chromatography (HPLC).
[0078] Purification and detection of natamycin products: the fermentation stock solution was centrifuged at 12000rpm for 10min to collect the precipitate, washed with pure water 2-3 times, and then 60% ethanol was added thereto at a ratio of 1:100 (M / V), and the pH was adjusted to 10.5-11 until natamycin was dissolved, and then centrifuged again at 12000rpm for 10min to remove the insoluble matter of the bacteria, and the supernatant was collected. The pH of the supernatant was adjusted to 6.5-7.0 until natamycin was precipitated, and after precipitation, the natamycin product was obtained by centrifugation again. The product was dissolved with the same volume of methanol, filtered through a 0.45μm membrane filter, and then its mass purity was detected by HPLC.
[0079] HPLC chromatographic conditions are: YMC-Pack pro C18, (250×4.6mm, 5μm) detection wavelength 303nm. Mobile phase A is 24% acetonitrile aqueous solution. In addition, 5mL tetrahydrofuran, 3g ammonium acetate and 1g ammonium chloride are added to each liter of mobile phase. After fully dissolved, filter through a 0.45μm membrane filter before use. Elution conditions are 0-35min 100% A.
[0080] Example 5: Effect of gene expression on natamycin-producing bacteria
[0081] The engineered bacteria NTIII-KDH and the control NTIII Streptomyces chrysoporus were fermented in shake flasks. After the fermentation, the fermentation stock solution was detected by HPLC ( Figure 4 ) and the contents of natamycin and the intermediates 4,5-de-epoxynatamycin (1) and 4,5-de-epoxy-desuganomycin (2) in the natamycin product treated by the above purification method (Table 1, Figure 5 ), the above experimental group was carried out in three groups in parallel, and the specific operation is shown in Example 4.
[0082] Table 1 Purity of natamycin and its intermediate compounds in natamycin products
[0083]
[0084] Table 2 Natamycin production of NTIII and recombinant bacteria NTIII-KDH
[0085] strains Yield (g / L) NTIII 13.0±0.4 NTIII-KDH 16.0±0.3
[0086] The results showed that the glycosyltransferase, P450 monooxygenase and efflux pump protein were introduced into Streptomyces NTIII, and the recombinant bacteria NTIII-KDH obtained had a natamycin yield of 16 g / L compared with the control, which was increased by 23% (Table 2); the fermentation stock solution was directly detected at the corresponding retention time and no accumulation of intermediates 4,5-deepoxy natamycin (1) and 4,5-deepoxy desaccharide natamycin (2) was observed, indicating that the intermediates were completely converted; at the same time, because the fermentation broth of the recombinant strain has a single main product, the subsequent purification operation can be simplified to a certain extent to obtain a product with higher purity. After treatment, the purity of natamycin was above 99%; in addition, the ability of the bacteria to transport natamycin to the extracellular space was improved, and the toxic pressure during the fermentation process was alleviated. In summary, the recombinant bacteria NTIII-KDH has excellent fermentation performance and is suitable for industrial large-scale fermentation production.
Claims
1. A recombinant Streptomyces chrysoporus for producing high-purity natamycin, characterized in that: The recombinant Streptomyces chrysogenum is obtained by introducing the glycosyltransferase encoding gene sgnk, the P450 monooxygenase encoding gene sgnd and the efflux pump protein encoding gene sgnh into the Streptomyces chrysogenum through a vector in combination with a promoter; The nucleotide sequence of the gene sgnk is shown as SEQ ID NO.1, the nucleotide sequence of the gene sgnd is shown as SEQ ID NO.2, and the nucleotide sequence of the gene sgnh is shown as SEQ ID NO.
3.
2. The recombinant Streptomyces chrysoporus according to claim 1, characterized in that The nucleotide sequence of the promoter is shown in SEQ ID NO.
4.
3. The method for constructing the recombinant Streptomyces chrysoporus for producing high-purity natamycin according to claim 1, characterized in that: This includes any of the following options: Scheme 1: The method for constructing the recombinant Streptomyces chrysoporus comprises the following steps: (1) Using the genomic DNA of Streptomyces chrysogenum as a template, PCR amplification was performed to obtain the sgnk gene fragment, and using the pSET152 plasmid as a template, PCR amplification was performed to obtain the Pint promoter fragment. After the two fragments were purified, they were spliced with the pMS82 linear vector. The spliced products were transformed into Escherichia coli. After verification, the obtained transformants were extracted to obtain the pMS82-Pint-sgnk plasmid; The nucleotide sequence of the Pint promoter fragment is shown in SEQ ID NO.4; (2) Obtain pMS82-Pint-sgnd and pMS82-Pint-sgnh plasmids using the same method as step (1); (3) The pMS82-Pint-sgnk plasmid was digested with MfeI and SpeI to obtain the Pint-sgnk-Mfe / Spe fragment, and the pMS82-Pint-sgnd plasmid was digested with XbaI and PstI to obtain the Pint-sgnd-Xba / Pst fragment. The obtained fragments Pint-sgnk-Mfe / Spe and Pint-sgnd-Xba / Pst were enzymatically linked with pMS82-Mfe / Pst, and the ligation system was transformed into Escherichia coli. After verification, the obtained transformants were extracted to obtain the pMS82-Pint-sgnk-Pint-sgnd plasmid; (4) The pMS82-Pint-sgnk-Pint-sgnd plasmid was digested with MfeI and SpeI to obtain the Pint-sgnk-Pint-sgnd-Mfe / Spe fragment, and the pMS82-Pint-sgnh plasmid was digested with XbaI and PstI to obtain the Pint-sgnh-Xba / Pst fragment. The two fragments were ligated with the pMS82-Mfe / Pst enzyme, and the ligation system was transformed into Escherichia coli. After verification, the obtained transformants were extracted to obtain the pMS82-Pint-sgnk-Pint-sgnd-Pint-sgnh plasmid; (5) Transform the plasmid obtained in step (4) into the competent medium of ET12567 / pUZ8002 to obtain the donor bacteria ET12567 / pUZ8002 / pMS82-Pint-sgnk-Pint-sgnd-Pint-sgnh; (6) performing conjugation transfer between the donor bacteria obtained in step (5) and Streptomyces chrysoporus to obtain a conjugate, and after purification and PCR verification of the conjugate, obtaining a recombinant Streptomyces chrysoporus; Scheme 2: The method for constructing the recombinant Streptomyces chrysoporus comprises the following steps: ① The gene sgnk, gene sgnd, gene sgnh and Pint promoter were biosynthesized to obtain the Pint-sgnk-Pint-sgnd-Pint-sgnh long fragment, which was spliced with the pMS82 linear vector by seamless cloning, and the splicing system was transformed into Escherichia coli. After verification, the obtained transformants were extracted to obtain the pMS82-Pint-sgnk-Pint-sgnd-Pint-sgnh plasmid; The nucleotide sequence of the Pint promoter fragment is shown in SEQ ID NO.4; ②Transform the plasmid obtained in step ① into the competent medium of ET12567 / pUZ8002 to obtain the donor bacteria ET12567 / pUZ8002 / pMS82-Pint-sgnk-Pint-sgnd-Pint-sgnh; ③ The donor bacteria obtained in step ② are conjugated with Streptomyces chrysoporus to obtain conjugates, and the conjugates are purified and PCR verified to obtain recombinant Streptomyces chrysoporus.
4. A Streptomyces sp. NTIII-KDH, deposited on October 28, 2024, deposited by China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20242353, and a deposit address of Wuhan University, Wuhan, China.
5. Use of the recombinant Streptomyces chrysoporus according to claim 1, the recombinant Streptomyces chrysoporus constructed by the method according to claim 3, or the Streptomyces chrysoporus NTIII-KDH according to claim 4 in the preparation of natamycin.
6. A method for preparing natamycin by the recombinant Streptomyces chrysoporus according to claim 1, the recombinant Streptomyces chrysoporus constructed by the method according to claim 3, or the Streptomyces chrysoporus NTIII-KDH according to claim 4, comprising the following steps: The Streptomyces chrysoporus is activated, then inoculated into a seed culture medium and cultured to a logarithmic phase, inoculated into a fermentation medium at a volume ratio of 1-1.5%, and fermented at 28-30° C. for 4-5 days to obtain a fermentation liquid, which is purified to obtain natamycin.
7. The method according to claim 6, characterized in that The components of the seed culture medium include: 2-4 g / L yeast powder, 4-6 g / L peptone, 4-6 g / L glucose, 2-6 g / L NaCl, pH 7.0-7.4, and the solvent is water.
8. The method according to claim 6, characterized in that The components of the fermentation medium include: 4-7 g / L yeast powder, 15-25 g / L peptone, 15-20 g / L glucose, 1-3 g / L NaCl, and the solvent is water.