Construction of an engineered bacterium for biosynthesis of disaccharidyl natamycin and application thereof

Abstract

The application discloses a kind of biosynthesis disaccharide natamin engineering bacteria construction and its application, belong to biotechnology field.The biosynthesis disaccharide natamin engineering bacteria of the application is the natamin streptomyces of heterologous expression nppY gene, by conjugation transfer nppY gene expression plasmid into natamin streptomyces, and resistance screening can obtain biosynthesis disaccharide natamin engineering bacteria.The water solubility of disaccharide natamin is increased by 107.6 times relative to natamin, hemolytic toxicity is reduced to 1 / 10 of natamin, and antifungal activity is 1 / 2 of natamin.The T value of disaccharide natamin is superior to natamin, and it can be used for preparing antifungal drug, and has good application prospect in antifungal aspect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to construction of an engineered bacterium for biosynthesis of disaccharide nata-mycin and application thereof. BACKGROUND

[0002] Nata-mycin belongs to the class of polyene macrolide antibiotics, which has excellent antifungal activity, but poor hemolytic and water-soluble properties greatly limit its clinical application. Therefore, some researchers solve this problem by preparing polyene antibiotic liposomes and chemical derivatives, and some researchers put their eyes on the modification of the biosynthetic pathway, hoping to synthesize polyene macrolide antibiotic derivatives with more comprehensive properties by modifying the carboxyl, glycosyl, hydroxyl or conjugated double bond on the macrocyclic backbone of PKS (polyketide synthase) post-modification.

[0003] NPP A1, 67-121C and nystatin P1 are representatives of natural disaccharide polyene macrolides. Among them, NPP A1 is a nystatin containing an additional N-acetylglucosamine, which is found in a strain of Pseudomonas autotrophica. Kim et al. identified the biosynthetic genes of NPP A1 by comparing with the biosynthetic genes of nystatin, and then they identified the molecular weight and chemical structure of the compound by HPLC-MS and NMR methods. Lee et al. further studied the properties of the compound and found that the water solubility of NPP A1 is 300 times that of nystatin, the hemolytic toxicity is 1 / 10 of that of nystatin, and the antifungal activity is 1 / 2 of that of nystatin. In order to understand the synthesis mechanism of the compound, Kim et al. identified the gene nppY encoding the second glycosyltransferase, which has 51% identity with the second glycosyltransferase gene pegA of 67-121C, and also determined that the addition of the second sugar group is also prior to the hydroxylation step catalyzed by NppL. Subsequently, Stephens et al. determined that the active site of NppY is arginine at position 200. Barke et al. found that the disaccharide nystatin is nystatin P1, which contains a second glycosyltransferase NypY in the biosynthetic gene cluster, which has 41% identity with NypD I. Poire et al. integrated the nypY gene into a strain producing amphotericin B and found that it can add a second sugar group to about 5% of the polyene substances, and cannot add a second sugar group to amphotericin without trehalosamine or carboxyl, in addition, they also determined that NypY and PegA two enzymes can recognize candicidin. Walmsley et al. greatly improved the yield of polyene macrolide antibiotics adding a second sugar group, and they found that if 8-deoxyamphotericin B is used as a substrate, the conversion efficiency of NypY can be increased to 40%.

[0004] The chemical structure of the natamycin molecule can be summarized as a macrocyclic lactone with a hydrophobic head and a side chain chromophore, and a hydrophilic tail and another side polyol region. The hemiketal structure of the tail can maintain the stereochemical configuration of the natamycin molecule, which can provide protection for the biological activity of natamycin, while the conjugated double bond of the chromophore region can provide rigidity to the stereochemical configuration of the molecule, and facilitate the ordered arrangement of the hydroxyl and epoxy groups in the polyol region. The structural characteristics of enol and ketone determine the characteristics of low water solubility and easy formation of micro-aggregate in aqueous solution of natamycin, which leads to uneven distribution of natamycin in tissues and organs and is not easy to be metabolized, so in clinical application, natamycin is mostly an external medicine rather than an injection or oral medicine. Therefore, the synthesis of natamycin derivatives with high water solubility will greatly improve its medicinal value. SUMMARY

[0005] The purpose of the present application is to solve the shortcomings and deficiencies of the prior art, and to provide a natamycin derivative disaccharide natamycin, a streptomyces strain capable of biosynthesizing disaccharide natamycin and a construction method thereof, and the application of the derivative disaccharide natamycin.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] A natamycin derivative is a disaccharide natamycin with the following structure.

[0008]

[0009] A streptomyces engineering strain for biosynthesizing disaccharide natamycin is a natamycin-producing streptomyces heterologously expressing nppY gene. Further, the natamycin-producing streptomyces is S. gilvosporeus.

[0010] The construction method of the streptomyces engineering strain for biosynthesizing disaccharide natamycin comprises the following steps: constructing an nppY gene expression plasmid, transferring the nppY gene expression plasmid into the natamycin-producing streptomyces by conjugation, and obtaining the natamycin-producing streptomyces heterologously expressing nppY gene through resistance screening, i.e. obtaining the streptomyces engineering strain for biosynthesizing disaccharide natamycin.

[0011] The above-mentioned streptomyces engineering strain for biosynthesizing disaccharide natamycin is applied in the production of disaccharide natamycin.

[0012] A method for producing disaccharide natamycin comprises the following steps: inoculating the streptomyces engineering strain for biosynthesizing disaccharide natamycin into a fermentation medium for fermentation to obtain a fermentation product containing disaccharide natamycin. The fermentation conditions are preferably 28°C for 102-108h.

[0013] Furthermore, the method for producing disaccharide-based natamycin also includes separation and purification of the disaccharide-based natamycin, and the separation and purification method includes the following steps:

[0014] (1) The fermentation product was centrifuged to retain the supernatant, and the precipitate was extracted with methanol.

[0015] (2) The precipitated extract and the supernatant collected in step (1) are thoroughly mixed and centrifuged again to collect the supernatant.

[0016] (3) Remove the methanol from the supernatant.

[0017] (4) After the remaining solution was allowed to stand for a long time, the supernatant was collected by centrifugation and the supernatant was collected by C 18 Separation was performed by reverse phase chromatography.

[0018] The water solubility of the disaccharide natamycin of the present invention is increased by 107.6 times compared with natamycin, the hemolytic toxicity is reduced to 1 / 10 of that of natamycin, the antifungal activity is 1 / 2 of that of natamycin, and the T value (comprehensive property score) of the disaccharide natamycin is better than that of natamycin. The disaccharide natamycin can be used to prepare antifungal drugs.

[0019] An antifungal drug containing disaccharide natamycin.

[0020] Advantages and beneficial effects of the present invention: An engineered Streptomyces strain capable of biosynthesizing disaccharide-natamycin was successfully constructed, yielding a natamycin derivative with improved water solubility. The method provided by the present invention enables the production of disaccharide-natamycin with a purity exceeding 95%. The T value (comprehensive property score) of disaccharide-natamycin is superior to that of natamycin, suggesting promising antifungal applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the construction of pSPU214-nppY plasmid.

[0022] Figure 2 Schematic diagram of the construction of pSET152-nppY plasmid.

[0023] Figure 3 HPLC analysis of fermentation products of S.gilvosporeus and S.gilvosporeus:nppY. A: HPLC analysis of S.gilvosporeus fermentation products; B: HPLC analysis of S.gilvosporeus:nppY fermentation products; C: Spectral absorption characteristics of each S.gilvosporeus:nppY product; D: Comparison of the optical absorption curves of the new product and natamycin. 1: A highly polar byproduct, 2: Natamycin, and 3: The new product.

[0024] Figure 4 Figure 8 is a HPLC analysis of the fermentation product of S. noursei ΔnysR IV and S. noursei ΔnysR IV expressing nppY. The upper side corresponds to the fermentation product of S. noursei ΔnysR IV and the lower side to the fermentation product of S. noursei ΔnysR IV expressing the nppY gene; the increase in the area of the UV absorption peak at 2 min indicates an increase in the large polar by-product; 1 is the UV absorption peak of the production of tetramycin B; 2 is the UV absorption peak of the production of tetramycin A.

[0025] Figure 5 Figure 9 is a HPLC analysis of the fermentation product of S. noursei S91-S and S. noursei S91-S expressing nppY. The upper side corresponds to the fermentation product of S. noursei S91-S and the lower side to the fermentation product of S. noursei S91-S expressing the nppY gene; the increase in the area of the UV absorption peak at 2 min indicates an increase in the large polar by-product; 1 is the UV absorption peak of the production of nystatin.

[0026] Figure 6 Figure 10 is a molar concentration curve of natamycin.

[0027] Figure 7 Figure 11 is the purification steps and HPLC analysis of the new fermentation product of S. gilvosporeus: nppY. A: purification steps; B: HPLC analysis of the sample after purification.

[0028] Figure 8 Figure 12 is the MS spectrum of the new fermentation product of S. gilvosporeus: nppY, disaccharidyl natamycin.

[0029] Figure 9 Figure 13 is the1H NMR spectrum of the new fermentation product of S. gilvosporeus: nppY, disaccharidyl natamycin. 1 H and 13 C NMR spectrum. A: 1 H NMR spectrum; B: 13 C NMR spectrum.

[0030] Figure 10 Figure 14 is the structure of the new fermentation product of S. gilvosporeus: nppY, disaccharidyl natamycin.

[0031] Figure 11 Figure 15 is the antibacterial activity of disaccharidyl natamycin and natamycin.

[0032] Figure 12 Figure 16 is the hemolytic activity of disaccharidyl natamycin and natamycin. A: supernatant of red blood cells lysed; B: hemolysis curve of natamycin; C: hemolysis curve of disaccharidyl natamycin.

[0033] Figure 13is the growth curve of S. gilvosporeus: nppY.

[0034] Figure 14 is the relationship between disaccharide natamycin yield and time and temperature of S. gilvosporeus: nppY fermentation. DETAILED DESCRIPTION

[0035] The following examples are intended to further illustrate the present application and are not intended to limit the same. Unless otherwise indicated, the techniques utilized in the examples are standard techniques commonly employed by those skilled in the art.

[0036] Example 1 Construction of disaccharide natamycin-producing strain and purification and identification of disaccharide natamycin

[0037] (1) Construction of nppY plasmid vector containing promoter and terminator

[0038] The nppY gene (the sequence is shown in SEQ ID NO. 1) was synthesized by Shengong Bioengineering Company and cloned between the Nco I and Hind III restriction sites of pUC57 plasmid. The kanM1 gene in pM1 plasmid (the construction of pM1 plasmid is described in the literature: Zheng Wu, Wenli Gao, Shaotong Zhou, Zhaolin Wen, Xianpu Ni, Huanzhang Xia*. Improving gentamicin Band gentamicin C1a production by engineering the glycosyltransferases that transfer primary metabolites into secondary metabolites biosynthesis. Microbiological Research, 2017, 203: 40-46.) was also cloned between the Nco I and Hind III restriction sites, and was connected with PhrdB promoter and t0 terminator. Figure 1

[0039] As shown in Figure 1 , the pM1 plasmid containing PhrdB promoter and t0 terminator and the pUC57 plasmid containing nppY gene were simultaneously digested with Nco I and Hind III, and the nppY gene was ligated into the pM1 vector from which the kanM1 gene was removed, so as to complete the construction of nppY plasmid vector pSPU241-nppY containing promoter and terminator.

[0040] ​The constructed pSPU241-nppY plasmid was verified by digestion with NcoI and HindⅢ. After electrophoresis, a 1442 bp nppY gene fragment was observed, proving that the obtained plasmid was correct.

[0041] (2) Construction of site-specific recombinant plasmid pSET152-nppY

[0042] The pSPU241-nppY plasmid was digested with BglII and the PhrdB-nppY-t0 fragment of about 2300 bp was recovered.

[0043] The plasmid pSET152 was digested with BamHI and the recovered product was dephosphorylated. The fragment containing the nppY gene was then ligated with the pSET152 vector using T4-DNA ligase to obtain the plasmid pSET152-nppY ( Figure 2 ).

[0044] The constructed pSET152-nppY plasmid was digested with NcoI and HindⅢ, and a 1442 bp nppY gene fragment was observed after electrophoresis, proving that the plasmid was constructed correctly.

[0045] (3) Construction of Streptomyces expressing nppY gene

[0046] The obtained pSET152-nppY plasmid was transformed into Escherichia coli ET12567 (pUZ8002) and Am R 、Cl R and Km R strains, and obtained transformant E. coli ET12567 (pUZ8002, pSET152-nppY).

[0047] E. coli ET12567 (pUZ8002, pSET152-nppY) as the donor strain, Streptomyces gilvosporeus ATCC13326 producing natamycin, Streptomyces hygroscopicus ΔnysRIV (ΔnysRIV is a positive regulatory gene nysRIV in Streptomyces hygroscopicus CGMCC 4.7082, which is knocked out to block nystatin production, so as to produce tetramycin), Streptomyces hygroscopicus S91-S (the construction of S91-S is described in the literature: Ren, J., Cui, Y., Zhang, F., Cui, H., Ni, X., Chen, F., Li, L., & Xia, H. (2014). Enhancement of nystatin production by redirecting precursor fluxes after disruption of the tetramycin gene from Streptomyces ahygroscopicus. Microbiological Research, 169(7-8), 602-608) as the recipient strain were used for conjugation transfer and Am R Strain screening, S. gilvosporeus: nppY, ΔnysRIV: nppY, S91-S: nppY which can express nppY gene were obtained.

[0048] (4) HPLC analysis of fermentation products

[0049] The above obtained Streptomyces expressing nppY gene was expanded in slant medium (20 g soluble starch, 0.5 g K2HPO4, 0.5 g MgSO4, 0.5 g NaCl, 1 g KNO3, 0.01 g FeSO4, 1 g beef extract, add distilled water to 1 L, add 1.5 g agar per 100 mL, pH value between 7.0-7.2) for 7-9 days, and then 1 cm 2 The expanded spores were cultured in seed medium (2 g glucose, 0.6 g fish peptone, 0.6 g yeast powder, 1 g NaCl, add distilled water to 100 mL, pH 7) at 28°C for 24-28 h, and 3 mL OD 600The bacteria liquid with OD600=4-6 was inoculated into 30 mL of fermentation medium (0.8 g corn starch, 2 g corn flour, 3 g glucose, 3 g soybean cake powder, 0.02 g NaCl, 0.02 g K2HPO4, 0.02 g MgSO4, 0.02 g FeSO4, 0.25 g (NH)2SO4, 0.5 g CaCO3 was added after adjusting pH to 7, 10% inoculation) and cultured at 28°C for 96 h for fermentation.

[0050] The fermentation product was centrifuged for 10 min (3500 r / m) to separate the supernatant and the precipitate, the supernatant was reserved and the precipitate was extracted with 10 mL of methanol for 30 min. The supernatant of the fermentation liquid and the precipitate extraction liquid were analyzed by using HPLC.

[0051] Compared with S. gilvosporeus, the fermentation product of S. gilvosporeus:nppY contained a new ultraviolet absorption peak (B, 8 min, labeled as 3), which indicated that S. gilvosporeus:nppY could ferment new products, and the polarity of the new product was greater than that of natamycin, and the spectral absorption characteristics of the new product were the same as those of natamycin (C, D). Figure 3 Compared with ΔnysR IV, the fermentation product of ΔnysR IV:nppY had no difference, and no new product was fermented (E). Figure 3 Compared with S91-S, the fermentation product of S91-S:nppY had no difference, and no new product was fermented (F). Figure 4 Figure 5 The spectral absorption characteristics of the new product were the same as those of natamycin, so the same amount of the new product and natamycin could show the same peak area in HPLC analysis, and the standard natamycin with gradient mass concentration was selected for HPLC analysis, and the ultraviolet absorption peak area was used to plot the mass concentration and fit (G), and the molar concentration curve applicable to both was drawn to facilitate subsequent analysis of the new product, and the peak area interval applicable to the curve was 337-37321.

[0052] The spectral absorption characteristics of the new product were the same as those of natamycin, so the same amount of the new product and natamycin could show the same peak area in HPLC analysis, and the standard natamycin with gradient mass concentration was selected for HPLC analysis, and the ultraviolet absorption peak area was used to plot the mass concentration and fit (G), and the molar concentration curve applicable to both was drawn to facilitate subsequent analysis of the new product, and the peak area interval applicable to the curve was 337-37321. Figure 6

[0053] (5) Purification of the new product

[0054] Several HPLC analyses of the supernatant and precipitate extraction of the fermentation liquid of S. gilvosporeus:nppY were performed, and it was found that the new product contained in the supernatant had a higher concentration.

[0055] ​​First step, S. gilvosporeus: nppY was inoculated into fermentation medium and cultured at 28°C for 96h for fermentation. The supernatant was reserved by centrifugation and the precipitate was extracted with methanol for 30min. Second step, the supernatant collected in the first step was mixed with the precipitate extract and centrifuged again. The supernatant was collected. Third step, the recovered supernatant was evaporated by rotary evaporation to remove methanol in the solvent. Fourth step, the remaining solution was left in the refrigerator at 4°C overnight. Fifth step, the supernatant was collected by centrifugation and analyzed by HPLC. Sixth step, the supernatant was collected by centrifugation and analyzed by HPLC. 18 The new product was separated by packed reverse phase chromatography (semi-preparative separation conditions: mobile phase: methanol: water (60:40); flow rate: 8 mL / min; C 18 reverse phase chromatography column; column temperature 30°C). Figure 7 A). The purity of the final separated sample could reach more than 95% Figure 7 B).

[0056] The yield of the new product by shake flask fermentation could reach 0.13g / L and the concentration of the new product after the above purification could reach at least 0.19mg / mL.

[0057] (6) Identification of the new product

[0058] The purified new product was collected and the water and methanol in the sample were removed by rotary evaporation and freeze-drying. The new product was analyzed by MS and the results showed that the molecular weight of [M+H] + and [M+Na] + was 869 and 891 respectively, which was consistent with the molecular weight of disaccharide-based natamycin. Figure 8 ).

[0059] Then deuterated methanol (CD3OD) was used for dissolution and NMR identification. The obtained 1 H and 13 C NMR spectra are shown in Figure 9 and the structure of the new product was determined as shown in Figure 10 and was named disaccharide-based natamycin.

[0060] Example 2 Property identification of disaccharide-based natamycin

[0061] (1) Water-soluble identification

[0062] A 10 mmol / L Tris-HCl solution was prepared, pH 7; 1 mg of disaccharide natamycin and 1 mg of natamycin were dissolved in 50 μL and 500 μL of the Tris-HCl solution, respectively; the solution was shaken thoroughly and saturated by ultrasonic treatment for 15 min; centrifugation was performed for 10 min (10000 r / min), and the supernatant was taken. To avoid the high concentration of the sample from being not completely eluted in the chromatographic column or the light absorption signal from exceeding the detection range of the cell, the disaccharide natamycin was diluted 50 times before being loaded. The solubility (S) of natamycin in water was calculated to be 34.4 μg / mL and the solubility (S) of disaccharide natamycin in water was calculated to be 3.7 mg / mL according to the HPLC analysis results and the molar concentration curve. According to the results, the water solubility of disaccharide natamycin was increased by 107.6 times compared with that of natamycin, indicating that the addition of the second sugar group greatly improved the water solubility of natamycin.

[0063] (2) Antifungal activity identification

[0064] The purified disaccharide natamycin was concentrated to 10.7 μg / mL and gradient diluted, and the concentration was reduced to half of the original concentration each time. The lowest concentration of the dilution was 0.1675 μg / mL. Saccharomyces cerevisiae was inoculated in a 96-well plate using 90 μL of yeast culture medium (2 g of peptone, 2 g of glucose, 1 g of yeast extract, and distilled water to make up to 100 mL) and 10 μL of the diluted antibiotic sample, 1000 CFU per well; incubation was performed at 28°C, and the absorbance at 490 nm was measured after 16 h. The antibiotic concentration that produced the inhibition was plotted against the reduced absorbance at 490 nm relative to the positive control, and a logarithmic trend line and standard equation were added. The lowest antibiotic concentration corresponding to the complete inhibition of Saccharomyces cerevisiae was the MIC value, and the MIC value when 50 / 90% of Saccharomyces cerevisiae was inhibited was estimated from the 50 / 90% of the growth inhibition curve. Three groups of inhibition experiments were simultaneously performed, and the inhibition data of disaccharide natamycin and natamycin were obtained according to the curve. The average value of the data was calculated, and the MIC value of disaccharide natamycin was 2.5 μg / mL, the MIC value was 1.79±0.02 μg / mL, the MIC value was 2.47±0.01 μg / mL, the MIC value of natamycin was 1.25 μg / mL, the MIC value was 0.77±0.05 μg / mL, and the MIC value was 1.14±0.02 μg / mL under the same conditions. Finally, the growth inhibition curves of disaccharide natamycin and natamycin were plotted according to the obtained values. 50 90 50 90 Figure 11

[0065] ​​​​​(3) Hemolytic activity identification

[0066] After extracting red blood cells from human plasma, they were lysed with physiological saline to a final red blood cell concentration of 2.5%. Hemolytic activity was then tested using natamycin at a final concentration of 5 to 180 μg / mL and disaccharide natamycin at a final concentration of 78 to 2500 μg / mL, respectively. The absorbance was measured using a microplate reader. The experimental method is as follows:

[0067] Blood samples were collected and 1–2 mL of 0.1–0.2 mol / L EDTA was added to 1 mL of blood for anticoagulation.

[0068] The anticoagulated blood was centrifuged for 10 min (3000 × g) and the supernatant was discarded;

[0069] Add an equal volume of normal saline or PBS buffer and stir gently until mixed;

[0070] Centrifuge for 10 min (3000 × g) and discard the supernatant;

[0071] Repeat the previous step to completely remove white blood cells and platelets until the supernatant is transparent, indicating that the red blood cells have been washed clean (similar to DNA extraction), and the remaining sediment is the desired red blood cells;

[0072] Prepare a 5 mg / mL antibiotic stock solution in DMSO;

[0073] Dilute to 10-2000 μg / mL respectively;

[0074] Take 0.1 mL and mix it with 0.9 mL of saline suspension containing 2.5% (22.5 μL) red blood cells and incubate in a 37°C water bath for 30 min (0.1 mL of DMSO is a negative control);

[0075] After centrifugation for 2 min (5000 r / min), the absorbance of the supernatant at 545 nm was measured (a physiological saline suspension containing 2.5% red blood cells was used as a positive control).

[0076] The hemolysis rate of antibiotics at corresponding concentrations was calculated according to the following formula.

[0077]

[0078] The supernatant of lysed red blood cells Figure 12 As shown in A, Table 1 corresponds to the concentration of each well sample. Then the hemolysis rate of the antibiotics at the corresponding concentration was calculated, and then the hemolysis rate curve of the two antibiotics was drawn using these data ( Figure 12 B, C), and calculated the HC of natamycin according to the standard equation 50 The value was 165±0.25μg / mL, and the HC of disaccharide natamycin50 The value was 2083±2.62μg / mL (three groups of hemolytic experiments were carried out at the same time and the average value of the lowest hemolytic activity was taken). The results showed that the HC of disaccharide natamycin 50 The value increased by 12.6 times, indicating that it has a great improvement in hemolytic activity.

[0079] Table 1 Antibiotic concentrations corresponding to hemolysis experiments

[0080]

[0081] (4) Comprehensive evaluation

[0082] After measuring the changes in water solubility, antifungal activity and hemolytic activity of disaccharide-based natamycin, their comprehensive properties were evaluated, and the in vitro therapeutic index calculation formula of antibiotics mentioned by Won et al. (MIC / HC) was used to calculate the therapeutic index of the antibiotics. 50 ), and according to the actual situation, the formula is supplemented as follows:

[0083]

[0084] The obtained MIC and HC 50 Substitute the T value (solubility in water) into formula 2.2 to calculate and compare their comprehensive performance (the smaller the comprehensive property score T value, the better the comprehensive performance).

[0085] Antibiotics with good comprehensive properties are characterized by small MIC values, HC 50 The T value is large and the S value is large, that is, the smaller the T value in the formula calculation result, the better the overall properties of the antibiotic. The calculated T value of disaccharide natamycin is 0.001876, and the T value of natamycin is 0.04391. Therefore, the conclusion is that the overall properties of disaccharide natamycin are far superior to those of natamycin.

[0086] Example 3 Analysis of Disaccharidosyl Natamycin-producing Strains

[0087] (1) Analysis of strain growth status

[0088] The seed growth curve of the disaccharide-based natamycin-producing strain was statistically analyzed. 12 bottles of S.gilvosporeus:nppY spores were inoculated into the seed culture medium and cultured at 28°C for 72 hours. From the 12th hour onwards, the seed wet weight (S.gilvosporeus) was sampled and measured every 6 hours as a control. The results showed that the seed growth rate was low before 12 hours, the cell number increased rapidly during the logarithmic growth period from 12 to 36 hours, the cell number began to increase slowly during the stationary growth period from 36 to 42 hours, and the cell number began to decrease after 54 hours into the decline period. Therefore, the seed quality of 42 to 48 hours was the best and most suitable for fermentation culture ( Figure 13Compared with the starting strain, although the growth curve morphology did not change, the logarithmic growth phase of the starting strain was 6 to 30 hours, the steady growth phase was 30 to 36 hours, and the cells began to die after 48 hours. Its optimal seed state was 36 to 42 hours. In other words, the growth cycle of S. gilvosporeus:nppY was delayed by 6 hours compared with the starting strain. In addition, the cell growth activity was weaker than that of the starting strain, and the cell number was less than that of the starting strain.

[0089] (2) Disaccharide-based natamycin conversion efficiency

[0090] After S.gilvosporeus:nppY fermentation was complete, 20 μL of the supernatant and precipitate (containing the cells, culture medium, and metabolites) extracts were analyzed by HPLC. Based on the concentration curves, it was calculated that the disaccharide-containing natamycin concentration in the fermentation supernatant accounted for approximately 41% of the total antibiotic concentration, while that in the precipitate extract accounted for approximately 33%. Overall, the concentration of disaccharide-containing natamycin that S.gilvosporeus:nppY could convert accounted for approximately 38% of the total antibiotic concentration (conversion efficiency was unaffected by fermentation temperature and time).

[0091] (3) Distribution and yield analysis of disaccharide-based natamycin

[0092] S. gilvosporeus:nppY was fermented at 28°C or 30°C for 96-120 hours to analyze the production of disaccharide-based natamycin. HPLC analysis showed that temperature and fermentation time did not affect the distribution of disaccharide-based natamycin in the supernatant and precipitate of the fermentation broth. Calculations revealed that the concentration of disaccharide-based natamycin in the supernatant of each bottle of culture medium was approximately twice that of the precipitate. Figure 14 ).

[0093] Furthermore, statistical analysis of disaccharide-based natamycin production under different conditions revealed that temperature significantly impacted disaccharide-based natamycin production. S. gilvosporeus:nppY fermented at 30°C produced less disaccharide-based natamycin than at 28°C, indicating that 28°C was the optimal fermentation temperature for S. gilvosporeus:nppY. Regarding the fermentation endpoint, antibiotic production at both temperatures increased initially and then decreased within 96 to 120 hours, with peak production occurring between 102 and 108 hours. This interval was therefore chosen as the fermentation endpoint.

[0094] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An engineered Streptomyces bacterium for biosynthesizing disaccharide-based natamycin, characterized by: The Streptomyces engineering bacteria is heterologous expression npp Y gene-producing Streptomyces natamycin; the structure of the disaccharide natamycin is as follows: 。 2. The Streptomyces engineered bacterium according to claim 1, characterized in that: The natamycin-producing Streptomyces is Streptomyces chrysoporus.

3. The method for constructing the Streptomyces engineering bacteria according to claim 1 or 2, characterized in that: The following steps are included: Build npp Y gene expression plasmid, transferred by conjugation npp The Y gene expression plasmid was transformed into natamycin-producing Streptomyces, and heterologous expression was obtained after resistance screening. npp Y gene-producing Streptomyces natamycin.

4. Use of the engineered Streptomyces bacteria according to claim 1 or 2 in the production of the disaccharide natamycin according to claim 1.

5. A method for producing the disaccharide syl natamycin according to claim 1, characterized in that: The method comprises the following steps: inoculating the engineered Streptomyces bacteria seeds according to claim 1 or 2 into a fermentation medium for fermentation to obtain a fermentation product containing disaccharide natamycin.

6. The method according to claim 5, characterized in that: The fermentation condition is 28° C. for 102 to 108 hours.

7. The method according to claim 5, characterized in that: The invention also includes the separation and purification of disaccharide-based natamycin, wherein the separation and purification method comprises the following steps: (1) The fermentation product was centrifuged to retain the supernatant, and the precipitate was extracted with methanol; (2) The precipitated extract and the supernatant collected in step (1) are thoroughly mixed and centrifuged again to collect the supernatant; (3) removing methanol from the supernatant; (4) After the remaining solution is allowed to stand for a long time, centrifuge and collect the supernatant. 18 Separation was performed by reverse phase chromatography.

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