CN115386511A - Streptomyces tsukubaensis ZJXM0001 with high tacrolimus yield and low ascomycin yield and application thereof - Google Patents

Streptomyces tsukubaensis ZJXM0001 with high tacrolimus yield and low ascomycin yield and application thereof Download PDF

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CN115386511A
CN115386511A CN202210913412.9A CN202210913412A CN115386511A CN 115386511 A CN115386511 A CN 115386511A CN 202210913412 A CN202210913412 A CN 202210913412A CN 115386511 A CN115386511 A CN 115386511A
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zjxm0001
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金利群
邢明林
鲁笛
汪贤文
柳志强
郑裕国
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Zhejiang University of Technology ZJUT
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Abstract

The invention discloses streptomyces tsukubaensis ZJXM0001 with high tacrolimus yield and low ascomycin yield and application thereof, wherein the streptomyces tsukubaensis ZJXM0001 is inoculated to a fermentation culture medium and cultured for 144-192h at 26-30 ℃ and 180-240rpm to obtain fermentation liquor containing tacrolimus, and the fermentation liquor is separated and purified to obtain tacrolimus; the invention obtains a streptomyces tsukubaensis ZJXM0001 strain which can produce tacrolimus in high yield and can obviously reduce ascomycin as a byproduct by using UV and ARTP mutagenesis and resistance screening methods; compared with the original streptomyces tsukubaensis strain S1, the yield of tacrolimus of the streptomyces tsukubaensis ZJXM0001 strain is increased by 236% at the temperature of 28 ℃, the rotation speed of 200rpm and the culture time of 180h, the yield of the tacrolimus of the streptomyces tsukubaensis ZJXM0001 strain is increased by 166% compared with the original strain under the condition of the temperature of 26 ℃ and no other conditions, and the method has important significance for subsequent purification, extraction and industrialization.

Description

Streptomyces tsukubaensis ZJXM0001 with high tacrolimus yield and low ascomycin yield and application thereof
(I) the technical field
The invention relates to streptomyces tsukubaensis capable of producing tacrolimus at a high yield and ascomycin at a low yield and application thereof.
(II) background of the invention
Tacrolimus (FK 506) is a 23-membered ring macrolide antibiotic with the molecular formula of C 44 H 69 NO 12 The relative molecular mass is 804.02, the structure is shown in figure 1, the product is colorless crystal at normal temperature, is insoluble in water, and is soluble in organic solvents such as methanol, ethanol, ethyl acetate, diethyl ether, chloroform, acetone, etc. The melting point is 127-129 ℃, and the stability is higher under different storage conditions. Isolated from fermentation broth of Streptomyces tsukubaensis (Streptomyces tsukubaensis) found in soil of Osaka Fabo area in 1984 by Kumaozi pharmaceutical Co. FK506 is an immunosuppressant widely used in clinic, which has an action mechanism of inhibiting the production of lymphokines which enable various types of cells to interact in immune response, and is widely used for preventing rejection of transplanted organs and treating autoimmune diseases such as inflammatory skin diseases and eczema. Cyclosporine a (CsA) is commonly used clinically as an immunosuppressant to treat rejection after organ transplantation before tacrolimus is found. CsA is also a CaN inhibitor, the mechanism of immunosuppression is similar to tacrolimus, but the effect of tacrolimus is about 100 times that of CsA, FK506 gradually replaces the use of cyclosporine since FDA approval in 1994, and is now one of the best-selling drugs for treating solid organ transplant patients in the world. In addition, its neuroprotective and regenerative functions have attracted considerable attention for FK506 research. In recent years, FK506 has been sold in the immunosuppressant market in more than 20% of the world.
There are two main methods for tacrolimus production: chemical synthesis method and microbial fermentation method. The synthetic route for synthesizing the tacrolimus by the chemical method is long, the number of byproducts is large, the separation and purification are difficult, the cost of raw materials is high, and the industrial prospect is small.
The microbial fermentation method has the advantages of simple process flow, cheap and easily-obtained substrate and the like, the main byproduct in the fermentation method production is ascomycin, the proportion of the ascomycin in part of high-yield strains can reach fifty percent of that of the product, and the separation cost is greatly improved due to the extremely similar structure with tacrolimus, so that the strain which is dedicated to obtaining high-yield tacrolimus and simultaneously has low-yield ascomycin is an important exploration direction of the fermentation method.
Streptomycin is an aminoglycoside antibiotic that acts on the ribosomal 30S subunit to inhibit mRNA translation initiation, ultimately affecting bacterial growth. Research shows that under low-concentration magnesium ions, the stability of the ribosome 70S complex is improved, the level of ribosome circulating factors closely related to the dissociation of ribosome mRNA and tRNA termination complex is increased, abnormal protein synthesis activity of the mutant strain occurs in a growth stable period, and finally, the overexpression of antibiotics is triggered, so that the yield of tacrolimus in streptomyces tsukubaensis is improved.
Malic acid is white crystal or crystalline powder which is easily dissolved in water and ethanol, the knockout of phosphoenolpyruvate carboxylase (ppc) has obvious effect of improving the yield of tacrolimus, and the malic acid has stronger inhibition effect on the ppc, so the malic acid is used as selective pressure to inhibit the activity of the ppc.
The method mainly combines UV and ARTP, but rarely searches for improving the mutagenesis rate by selecting selective pressure, and based on the principle, the invention screens the mutagenized strain by using streptomycin and malic acid as selective pressure through research to finally obtain the streptomyces tsukubaensis with high tacrolimus yield and low ascomycin yield.
Disclosure of the invention
The invention aims to obtain a streptomyces tsukubaensis ZJXM0001 strain with high tacrolimus yield and low ascomycin yield by mutagenesis combined with resistance screening and application thereof in tacrolimus synthesis, thereby avoiding the phenomenon that a byproduct ascomycin is greatly increased while the tacrolimus is high in yield, and reducing the cost for subsequent purification of tacrolimus.
The technical method adopted by the invention is as follows:
the invention provides a new strain with high tacrolimus yield and low ascomycin yield, namely Streptomyces tsuknbaenis ZJXM0001 (Streptomyces tsuknbaenis ZJXM 0001), which is preserved in China center for type culture collection with the preservation date of 2021, 11 and 15 days and the preservation number of CCTCC NO: m20211418, wherein the preservation address is Wuhan university, wuhan, china, zip code 430072.
The streptomyces tsukubaensis ZJXM0001 with high tacrolimus yield and low ascomycin yield is obtained by mutagenesis and screening, and the method comprises the following steps:
(1) Inoculating wild streptomyces tsukubaensis S1 seed liquid to a GYM flat plate, culturing for 6-8 days at 26-30 ℃, eluting spores on the flat plate by using normal saline, filtering spore suspension, eluting the spores once by using the normal saline, centrifuging to remove supernatant, and resuspending by using the normal saline to obtain the spore suspension;
(2) Diluting the spore suspension obtained in the step (1) to a concentration of 10 6 ～10 8 Taking the diluted spore suspension, placing the diluted spore suspension under an ultraviolet lamp tube for irradiating for 45-120 s at a position of 20-30 cm (preferably irradiating for 90s at a position of 20 cm), taking the irradiated bacterial liquid, coating the irradiated bacterial liquid on a GYM solid culture medium containing 70 mu g/mL streptomycin and 0.3mmol/L malic acid, and culturing at 26-30 ℃ for 5-8 days until a single colony can be observed; after the obtained single colony is fermented, detecting the output of tacrolimus and ascomycin by liquid phase, and screening out a mutant strain with high tacrolimus yield and low ascomycin yield;
(3) Preparing spore suspension from the mutant strain according to the method in the step (1), treating the spore suspension for 50-60 s (preferably for 60s at 10 ℃ and 10L/min ventilation) by using an ARTP mutagen (ARTP-M, qingtian wood biological technology Co., ltd. Without a tin source) at 10-20 ℃ and 10-20L/min working gas flow, coating the treated spore suspension on a GYM solid culture medium containing 70 mu g/mL streptomycin and 0.3mmol/L malic acid, and culturing the spore suspension at 26-30 ℃ in a dark place; and (5) repeating the step (2) and the step (3), and screening to obtain the streptomyces tsukubaensis mutant strain with high tacrolimus yield and low by-product ascomycin yield.
The invention also provides the wave building chainThe application of the mould ZJXM0001 in preparation of tacrolimus through microbial fermentation is as follows: inoculating streptomyces tsukubaensis ZJXM0001 to a fermentation culture medium, culturing at 26-30 ℃ and 180-240rpm for 144-192h (preferably fermenting at 28 ℃ and 220rpm for 168 h) to obtain fermentation liquor containing tacrolimus with less ascomycin byproducts, and separating and purifying the fermentation liquor to obtain tacrolimus; the final concentration of the fermentation medium comprises: 40-60 g/L of starch, 30-50 g/L of dextrin, 5-20 g/L of glucose, 5-20 g/L of yeast powder, 1-5 g/L of peptone, (NH) 4 ) 2 SO 2 0.5～1.5g/L，CaCO 3 1～10g/L， K 2 HPO 4 0.5～1.5g/L，MgSO 4 0.1-1 g/L, water as solvent and pH7.0.
The preferred final concentration composition of the fermentation medium is: 55g/L of starch, 45g/L of dextrin, 10g/L of glucose, 15g/L of yeast powder, 3g/L of peptone, (NH) 4 ) 2 SO 4 0.7g/L，CaCO 3 8g/L，MgSO 4 0.8g/L， K 2 HPO 4 0.8g/L, solvent is water (distilled water or tap water), and pH is 7.0.
Before the fermentation of the streptomyces tsukubaensis ZJXM0001, firstly inoculating the streptomyces tsukubaensis ZJXM0001 to a seed culture medium, culturing to obtain seed liquid, and then inoculating the seed liquid to the fermentation culture medium according to the inoculation amount with the volume concentration of 5-15% (preferably 10%) for fermentation culture, wherein the seed culture process comprises the following steps: inoculating Streptomyces tsukubaensis ZJXM0001 to a GYM solid culture medium, and culturing at 28 deg.C for 4-7 days; selecting a single colony, inoculating the single colony into a seed culture medium, and culturing at 28 ℃ and 220rpm for 48 hours to obtain a seed solution; the seed culture medium comprises the following components: 20 to 35g/L of glycerol, 5 to 15g/L of starch, 5 to 15g/L of glucose, 5 to 15g/L of yeast powder and CaCO 3 0.5-1.5 g/L, naCl 5-15 g/L, water as solvent and pH7.0. The preferred seed medium composition: 25g/L of glycerol, 10g/L of starch, 1.5g/L of glucose, 10g/L of yeast extract and CaCO 3 1g/L, solvent is water, pH7.0.
Figure RE-GDA0003917575130000041
Compared with the prior art, the invention has the following beneficial effects: the invention obtains a streptomyces tsukubaensis ZJXM0001 strain which can produce tacrolimus in high yield and can obviously reduce ascomycin as a byproduct by using UV and ARTP mutagenesis and resistance screening methods; compared with the streptomyces tsukubaensis strain S1, the yield of tacrolimus of the streptomyces tsukubaensis ZJXM0001 strain is improved by 63%, and the yield of the byproduct ascomycin is reduced by 100%. The method has important significance for subsequent purification, extraction and industrialization. When the strain is cultured for 48 hours at the temperature of 28 ℃ and the speed of 220rpm, the original strain S1 has the tacrolimus yield of 51.20mg/L, the ascomycin yield reaches 32.20mg/L, the mutant strain ZJXM0001 has the tacrolimus yield of 156.9mg/L, and the by-product ascomycin yield is only 7.52mg/L. Under the conditions of changing the temperature to 26 ℃ and keeping other culture conditions unchanged, the mutant strain ZJXM0001 tacrolimus yield reaches 136.2mg/L, and no ascomycin is generated.
(IV) description of the drawings
FIG. 1 is a graph of the lethality of the strains of example 1 at different UV mutagenesis times.
FIG. 2 is a graph showing the lethality of the strain in example 1 at various ARTP mutagenesis time.
FIG. 3 is a graph showing the biomass change of the strain before and after mutagenesis in example 4.
(V) detailed description of the preferred embodiments
The invention will now be described in one step with reference to specific examples, without limiting the scope of the invention thereto:
the experimental methods in the experimental examples are conventional methods unless otherwise specified.
The experimental materials used in the examples, unless otherwise specified, were all conventional biochemical reagents.
The culture medium used in the embodiment of the invention comprises the following components:
the seed culture medium comprises the following components: 25g/L of glycerin, 10g/L of starch, 1.5g/L of glucose, 10g/L of yeast extract and CaCO 3 1g/L, solvent is water, pH7.0.
The initial fermentation medium comprises: 50g/L of starch, 35g/L of dextrin, 8g/L of glucose, 15g/L of yeast powder, 1g/L of peptone, (NH) 4 ) 2 5O 4 1g/L，NaCl1g/L，CaCO 3 6g/L, solvent is water, and pH is 7.0.
The fermentation medium comprises the following components: 60g/L of starch, 45g/L of dextrin, 10g/L of glucose, 20g/L of yeast powder, 3g/L of peptone, (NH) 4 ) 2 SO 4 2g/L，MgSO 4 1g/L，CaCO 3 8g/L, the solvent is water (distilled water or tap water), the pH is 7.0, and the sterilization is carried out for 30min at 115 ℃.
Composition of GyM liquid medium: malt extract 10g/L, glucose 4g/L, yeast extract 4g/L, calcium carbonate 2g/L, solvent is water (distilled water or tap water), pH7.0. The GyM solid medium is prepared by adding 20g/L agar to a GyM liquid medium. Sterilizing at 115 ℃ for 30min, cooling the GYM solid culture medium to 50-65 ℃ when mutagenizing and screening bacteria, and adding streptomycin and malic acid which are subjected to membrane/sterilization to make the final concentrations of streptomycin 70 mu g/mL and malic acid 0.3mmol/L respectively.
Example 1 screening of wild-type Streptomyces tsukubaensis ZJXM0000
1. Screening of Strain S1
(1) Primary screening: adding 10g of soil around Hangzhou vegetable garden (Taoyingling No. 1 in West lake region of Hangzhou Zhejiang) into 90ml of sterile water, and shaking to form a soil solution for later use. Diluting the soil solution with normal saline 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 After the volume is doubled, the streptomyces tsukubaensis is evenly coated in a GYM solid culture medium containing 30 mu g/mL streptomycin, the streptomyces tsukubaensis is cultured for 3-5 days at 28 ℃, the characteristics of colony color, surface structure and the like are observed, the colony of the streptomyces tsukubaensis is spherical, the surface is rough and has wrinkles, the edge color is light pink, the central part is red, and the streptomyces tsukubaensis is screened out according to the characteristics of the colony of the strain.
(2) Re-screening: inoculating the streptomyces tsukubaensis strain screened from the soil in the step (1) into a seed liquid culture medium, and culturing for 48 hours at 28 ℃ to obtain a seed liquid; inoculating the seed solution into a 500mL shake flask filled with 50mL primary fermentation medium in an inoculation amount of 10% volume concentration, and culturing for 168h in a shaking table at 28 ℃ and 220r/min to obtain fermentation liquor. The amount of tacrolimus produced was measured by the HPLC method described in example 4, and a strain with a high yield was selected and designated as strain S1 as a mutagenic starting strain.
2. Identification of Strain S1
(1) Physiological and biochemical characteristics
Streaking the strain S1 glycerol tube on a GYM plate, culturing at 28 ℃ for 4-6d, and observing colony morphology. The bacterial colony of the bacterial strain S1 is large and thin, the surface of the bacterial colony is wrinkled and dry and is not easy to pick up, the edge of the bacterial colony is light pink, and the central part of the bacterial colony is red.
(2) Physiological and biochemical identification
Strain S1 was phenotyped using the Biolog (GENIII) automated microorganism identification system with 94 phenotypic tests, including 71 carbon source utilization assays and 23 chemosensitivity assays: the strain S1 was inoculated into Sub>A specified plate medium, incubated at Sub>A constant temperature of 33 ℃ for 2 days, washed off the cells on the plate with Sub>A sterile cotton swab, mixed with an inoculum (IF-A) to prepare Sub>A bacterial suspension, and adjusted to 91% T/IF-A by Sub>A turbidimeter. The bacterial suspensions were added individually to each well of a Biolog GENIII microwell assay plate using an 8-well electric applicator, 100 μ L per well. Placing the micropore identification plate in an incubator at 33 ℃, respectively culturing for 12h, 24h and 36h, then placing the plate on a Biolog reading instrument to read results, and finally analyzing metabolic fingerprints by the Biolog reading instrument, wherein the strain S1 can strongly utilize 20 carbon sources, and cannot or has weak utilization capacity for other 51 carbon sources; the strain S1 is sensitive to 5 chemical substances. The results are shown in tables 1 and 2.
TABLE 1 ability of the Strain S1 to utilize 71 carbon sources
Figure BDA0003761864760000061
Figure BDA0003761864760000071
TABLE 2 chemosensitivity of strain S1 to 23 chemicals on Biolog GENIII plates
Figure BDA0003761864760000072
According to physiological and biochemical characteristics, the strain S1 is identified as Streptomyces tsuknbaenis (Streptomyces tsuknbainis), named as Streptomyces tsuknubaenis (Streptomyces tsuknbaenis) ZJXM0000, deposited in China center for type culture collection with the preservation date of 2022, 5 and 23 months and the preservation number of CCTCC NO: m2022704, sagnac Address Wuhan, wuhan university, china, post code 430072.
Example 2 selection of UV mutagenesis time
1. Preparation of spore suspension
The Streptomyces tsukubonensis ZJXM0000 (Streptomyces tsuknbaensis ZJXM 0000) screened in example 1 is used as a starting strain.
Streptomyces tsukubaensis ZJXM0000 is inoculated to a GYM solid plate, cultured for 7 days at 28 ℃, and eluted by 10mL of physiological saline to collect spore suspension. Filtering the washed spore suspension by using a syringe containing gauze, centrifuging the filtered spore at 8000rpm for 5min, removing supernatant, adding 10mL of physiological saline for re-suspension, centrifuging at 8000rpm for 5min, re-eluting once, and re-suspending by using 5mL of physiological saline as wild type spore suspension.
2. Selection of UV mutagenesis time
Different ultraviolet irradiation time can generate different mutation rates and lethality rates, and the optimal irradiation time is a key step of mutagenesis.
Taking the wild type spore suspension prepared in the step 1, and diluting the wild type spore suspension to the concentration of 10 by using deionized water 7 Placing the cells/mL of the strain under an ultraviolet lamp (15W, 254nm) for 20cm, irradiating the cells for 30s, 45s, 60s, 75s, 90s and 120s respectively, then inoculating the cells to a GYM solid culture medium containing 70 mu g/mL streptomycin and 0.3mmol/L malic acid, culturing the cells at 28 ℃ for 4 days, counting the number of single colonies on a plate, and respectively calculating the lethality of different mutagenesis time, as shown in figure 1. Generally, the positive mutation rate is highest under the mutagenesis condition with about 80% of lethality, so the ultraviolet mutagenesis time is selected to be 90s.
Example 3 determination of optimal mutagenesis time of ARTP
Different irradiation times of ARTP produce different mutation rates and lethality rates, and the optimal irradiation time is a key step of mutagenesis.
mu.L of the wild-type spore suspension prepared in example 1 was mixed with an equal volume of 10% glycerol on a metal slide of an ARTP mutagen (ARTP-M, qingtianmu Biotech Co., ltd. Without a tin source), spread uniformly, and treated with the ARTP mutagen at 10 ℃ for 15s, 30s, 45s, 60s, 90s, and 120s at a ventilation rate of 10L/min. After the treatment, the entire metal slide was put into a 1.5mL EP tube containing 990. Mu.L of physiological saline and sufficiently shaken. mu.L of liquid from each of the above EP tubes was spread on a GYM solid medium containing 70. Mu.g/mL streptomycin and 0.3mmol/L malic acid, and cultured at 28 ℃ in the dark for 4 days, and the number of single colonies on the plate was counted to calculate the lethality at different mutagenesis times, respectively, as shown in FIG. 2. Generally, the positive mutation rate is highest under the mutagenesis condition of about 80% lethality, so that the ARTP mutagenesis time is selected to be 60s.
Example 4: composite mutagenesis of original strain streptomyces tsukubaensis ZJXM0000
1. Wild type spore suspension
Streptomyces tsukubaensis ZJXM0000 is inoculated to a GYM solid plate, cultured for 7 days at 28 ℃, and eluted by 10mL of physiological saline to collect spore suspension. Filtering the washed spore suspension by using a syringe with gauze inside, centrifuging the filtered spore at 8000rpm for 5min, removing the supernatant, adding 10mL of physiological saline for re-suspension, centrifuging at 8000rpm for 5min, re-eluting once, and re-suspending by using 5mL of physiological saline as the wild type spore suspension.
2. UV mutagenesis
Taking the wild type spore suspension prepared in the step 1, and diluting the wild type spore suspension to the concentration of 10 by using deionized water 7 Irradiating each/mL of the cells at 20cm under an ultraviolet lamp (15W, 254nm) for 90s, inoculating the cells to a GYM solid medium containing 70. Mu.g/mL streptomycin and 0.3mmol/L malic acid, and culturing at 28 ℃ for 4 days; selecting a single colony, inoculating the single colony to a seed culture medium, and culturing at 28 ℃ and 220rpm for 2 days; inoculating the seed solution into a fermentation culture medium in an inoculation amount with the volume concentration of 10%, culturing at 28 ℃ and 220rpm for 7 days, mixing the fermentation liquor with methanol in the same volume, centrifuging at 12000rpm for 10min, taking supernate to pass through a 0.22-micron membrane, detecting the yields of tacrolimus and ascomycin by adopting high performance liquid chromatography, and screening out a mutant strain with high tacrolimus yield and low ascomycin yield.
The conditions for detecting the output of the tacrolimus and the ascomycin by adopting the high performance liquid chromatography are as follows: a chromatographic column: c18 (4.6X 250mm,5 μm), column temperature 60 ℃, mobile phase A: mobile phase B = 65: 35 (v/v), flow rate 0.9mL/min, ascomycin peak time 18.5min, tacrolimus peak time 19min, tacrolimus and ascomycin concentration in the product were obtained according to peak area and corresponding standard curve.
Under the same conditions, tacrolimus and ascomycin standards (all purchased from aladin company) are respectively prepared into 200mg/L mother liquor by using methanol as a solvent, the mother liquor is respectively diluted into 50mg/L, 100mg/L and 150mg/L standard liquor by using methanol, and the liquid phase measurement is carried out on the 200mg/L standard liquor, so as to obtain a standard curve of the concentration and the peak area of a substance. The standard curve of ascomycin is Y =2027X +3059.8, and the standard curve of tacrolimus is Y =5.527X-58.167.
Mobile phase a was acetonitrile.
The mobile phase B is phosphoric acid: methanol: water = 0.001: 1: 6 (v/v/v).
3. ARTP mutagenesis
Inoculating mutant strain thalli obtained by ultraviolet mutagenesis in the step 2 to a seed culture medium, and culturing for 48 hours at 28 ℃ to obtain seed liquid; 200. Mu.L of the seed solution was spread on a GYM solid plate, cultured at 28 ℃ for 7 days, and the spore suspension was collected by eluting with 10mL of physiological saline. Filtering the washed spore suspension by using a syringe with cotton gauze, centrifuging the filtrate at 8000rpm for 5min, removing supernatant, adding 10mL of physiological saline for re-suspension, centrifuging at 8000rpm for 5min, re-eluting once, and re-suspending by using 5mL of physiological saline to obtain the spore suspension.
Mixing 5 μ L spore suspension and equal volume of 10% glycerol on ARTP mutagen (ARTP-M, QINGTIANMU Biotech Co., ltd.) metal slide, spreading uniformly, and treating with ARTP mutagen at 10 deg.C and 10L/min ventilation amount for 45s. After the treatment, the entire metal slide was put into a 1.5mL EP tube containing 990. Mu.L of physiological saline and sufficiently shaken. And (3) taking 100 mu L of liquid from each EP tube, coating the liquid on a GYM solid culture medium containing 70 mu g/mL streptomycin and 0.3mmol/L malic acid, culturing for 4 days at 28 ℃ in a dark place, counting the number of single colonies on a plate, and screening according to the lethality to obtain a mutant strain. Each experiment was performed in 3 replicates.
4. Repeated mutagenesis
And (4) repeating the ultraviolet mutagenesis in the step (1) and the ARTP mutagenesis in the step (2) for 4 times by screening the mutant strains with high tacrolimus yield and low ascomycin yield in the step (3), and finally screening to obtain the mutant strain with the tacrolimus yield of 156.9mg/L and the byproduct ascomycin of 7.52mg/L, and recording the mutant strain as a strain ZJXM0001.
5. Identification of Strain ZJXM0001
(1) Physiological and biochemical characteristics
Streaking the screened strain ZJXM0001 glycerol tube in the step 4 on a GyM plate, culturing at 28 ℃ for 4-6d, and observing colony morphology. The bacterial colony of the ZJXM0001 is large and thin, the surface of the bacterial colony is wrinkled, dry and difficult to pick up, the edge of the bacterial colony is light pink, and the central part of the bacterial colony is red.
(2) Physiological and biochemical identification
94 phenotypic tests were performed on the mutagenized strain ZJXM0001 using the Biolog (GENIII) automated microbial identification system, including 71 carbon source utilization assays and 23 chemosensitivity assays: inoculating the strain to Sub>A specific plate medium, culturing at constant temperature of 33 deg.C for 2 days, washing the thallus on the plate with Sub>A sterile cotton swab, mixing with an inoculum (IF-A) to obtain Sub>A suspension, and adjusting to 91% T/IF-A with Sub>A turbidimeter. The bacterial suspensions were added to each well of the Biolog GENIII microwell assay plate using an 8-well electropipettor, 100 μ L per well. Putting the micropore identification plate in an incubator at 33 ℃, respectively putting the plate on a Biolog reader to read the result after 12h, 24h and 36h of culture, and finally analyzing the metabolic fingerprint by the Biolog reader. The strain ZJXM0001 can strongly utilize 33 carbon sources, and cannot utilize other 38 carbon sources or has weak utilization capacity; the strain ZJXM0001 is sensitive to 3 chemical substances, and the result is shown in tables 3 and 4.
TABLE 3 ability of Strain ZJXM0001 to utilize 71 carbon sources
Figure BDA0003761864760000101
Figure BDA0003761864760000111
TABLE 4 chemosensitivity of strain SZJXM0001 to 23 chemicals on Biolog GENIII plate
Figure BDA0003761864760000112
According to physiological and biochemical characteristics, the strain ZJXM0001 is identified as Streptomyces tsuknubaenis (Streptomyces tsuknbaenis), named as Streptomyces tsuknubaenis (Streptomyces tsuknbaenis) ZJXM0001, deposited in China center for type culture collection with the preservation date of 2021 year, 11 months and 15 days and the preservation number of CCTCC NO: m20211418, wherein the preservation address is Wuhan, wuhan university, china, zip code 430072.
Example 5 Streptomyces tsukubaensis ZJXM0001 Shake flask fermentation Condition optimization
1. Temperature of fermentation
(1) Preparing a seed solution: the Streptomyces tsukubaensis ZJXM0001 screened in example 4 is streaked on a GyM plate, cultured for 4 days at 28 ℃, single colony is picked up and inoculated into a seed culture medium, and cultured for 48 hours at 220rpm at 28 ℃ to obtain a seed solution.
(2) Fermentation culture
1. Temperature of fermentation
And (2) shaking a flask with the specification of 500mL, filling 50mL of fermentation medium, inoculating the seed solution in the step (1) according to 10% of the liquid filling volume, and performing fermentation culture for 168 hours at the temperature of 26 ℃, the temperature of 28 ℃ and the temperature of 30 ℃ respectively at the rotating speed of 220rpm to obtain fermentation liquor. Mixing the fermentation liquor with methanol in the same volume, centrifuging at 12000rpm for 10min, taking supernate, passing the supernate through a 0.22-micron membrane, detecting the yields of tacrolimus and ascomycin by adopting the high performance liquid chromatography described in example 4, and measuring the yield of tacrolimus to 156.9mg/L and the yield of ascomycin to 0mg/L to 8.4mg/L; wherein the output of the tacrolimus is 156.9mg/L and the output of the ascomycin is 7.52mg/L at 28 ℃.
TABLE 5 Effect of fermentation temperature on product yield
26℃ 28℃ 30℃
Tacrolimus mg/L 136.2 156.9 148.3
Ascomycin mg/L 0 7.52 8.4
2. Rotation speed of fermentation
Changing 220rpm in the fermentation culture in the step 1 into 180rpm, 200rpm and 240rpm, and the other operations are the same, finally measuring that the yield of the tacrolimus is 141.6mg/L to 167.4mg/L and the yield of the ascomycin is 7.04mg/L to 9.22mg/L; wherein the highest output of tacrolimus is 167.4mg/L and the output of ascomycin is 9.22mg/L under 200 rpm.
TABLE 6 influence of fermentation rotation speed on product yield
180rpm 200rpm 240rpm
Tacrolimus mg/L 141.6 167.4 151.2
Ascomycin mg/L 7.04 9.22 7.35
3. Fermentation culture time
Changing the fermentation culture time of the step 1 into 156h, 180h and 192h, and carrying out the same operation, wherein the final measured tacrolimus yield is 148.40mg/L to 162.4mg/L, and the ascomycin yield is 6.56mg/L to 9.40mg/L; wherein under 180 hours, the output of tacrolimus is 162.4mg/L, and the output of ascomycin is 6.56mg/L.
TABLE 7 Effect of fermentation time on product yield
156h 180h 192h
Tacrolimus mg/L 148.4 162.4 159.7
Ascomycin mg/L 7.80 6.56 9.40
The yield of tacrolimus of streptomyces tsukubaensis ZJXM0001 at different temperatures, rotating speeds and culturing times is researched according to the steps, and the result shows that the yield of tacrolimus is 156.9mg/L and the yield of ascomycin is 7.52mg/L at the temperature of 28 ℃, the rotating speed of 220rpm and the culturing time of 168 hours; under the conditions of the temperature of 28 ℃, the rotating speed of 200rpm and the culture time of 168 hours, the output of tacrolimus is 167.40mg/L and the output of ascomycin is 9.22mg/L; under the conditions that the temperature is 28 ℃, the rotating speed is 220rpm and the culture time is 180 hours, the output of the tacrolimus is 162.4mg/L and the output of the ascomycin is 6.56mg/L; finally, under the conditions that the temperature is 28 ℃, the rotation speed is 200rpm and the culture time is 180 hours, the yield of the streptomyces tsukubaensis ZJXM0001 tacrolimus is 172.4mg/L, the yield of the ascomycin is 5.60mg/L and the culture condition is 26 ℃. The rotating speed is 220rpm, the culture time is 168h, the yield of tacrolimus is 136.2mg/L, and no ascomycin is generated.
Example 6: streptomyces tsukubaensis ZJXM0001 shake-flask fermentation for producing tacrolimus and low-yield (non-production) ascomycin
(1) Preparing a seed solution: the original strain Streptomyces tsukubaensis ZJXM0000 in example 1 and the selected Streptomyces tsubaensis ZJXM0001 in example 4 are streaked on a GyM plate, cultured for 4 days at 28 ℃, single colony is picked up and inoculated into a seed culture medium, and cultured for 48h at 28 ℃ and 220rpm to obtain a seed solution.
(2) Fermentation culture
And (2) shaking a flask with the specification of 500mL, filling 50mL of fermentation medium, inoculating the seed solution in the step (1) according to 10% of the liquid filling volume, and performing fermentation culture at 28 ℃ and 220rpm for 168 hours to obtain fermentation liquid.
(3) Determination of Biomass
The biomass is determined by dry weight method, which comprises the following steps: and 2mL of fermentation solution is taken in an EP tube (each EP tube is weighed in advance) every 12h in the fermentation process, the centrifugation is carried out for 10min at 12000rpm, the supernatant is discarded, the thalli are collected, the thalli are continuously washed for 2-3 times by using sterile normal saline, the supernatant is discarded, the EP tube with thalli precipitates is placed at 80 ℃ to be dried to constant weight, then the biomass of the thalli is weighed and calculated, and the biomass is integrally increased, as shown in figure 3.
(4) Determination of Tacrolimus production
And (3) mixing the fermentation liquor obtained in the step (2) with methanol with the same volume, centrifuging at 12000rpm for 10min, taking the supernatant, passing the supernatant through a 0.22-micron membrane, and detecting the contents of tacrolimus and ascomycin by using the high performance liquid chromatography described in example 4, wherein the result shows that the yield of the original strain streptomyces tsukubaensis ZJXM0000 tacrolimus is 51.20mg/L, the yield of ascomycin is 32.20mg/L, the yield of the mutant strain ZJXM0001 tacrolimus is 156.9mg/L, and the yield of the byproduct ascomycin is only 7.52mg/L. Under the condition of changing the temperature to 26 ℃ and other culture conditions, the mutant strain ZJXM0001 tacrolimus yield reaches 136.2mg/L, and no ascomycin is generated.

Claims (6)

1. The Streptomyces tsukubnbaenis ZJXM0001 (Streptomyces tsuknbaenis ZJXM 0001) with high tacrolimus yield and low ascomycin yield is preserved in China center for type culture collection (CCTCC NO) with the preservation number of CCTCC NO: m20211418, wherein the preservation address is Wuhan, wuhan university, china, zip code 430072.
2. The application of the streptomyces tsukubaensis ZJXM0001 in the preparation of tacrolimus through microbial fermentation according to claim 1.
3. The use according to claim 2, wherein said use is: inoculating streptomyces tsukubaensis ZJXM0001 to a fermentation culture medium, culturing at 26-30 ℃ and 180-240rpm for 144-192h to obtain a fermentation liquid containing tacrolimus, and separating and purifying the fermentation liquid to obtain tacrolimus; the final concentration of the fermentation medium is as follows: 40-60 g of starchPer liter, 30 to 50g/L of dextrin, 5 to 20g/L of glucose, 5 to 20g/L of yeast powder, 1 to 5g/L of peptone and (NH) 4 ) 2 SO 2 0.5～1.5g/L，CaCO 3 1～10g/L，K 2 HPO 4 0.5～1.5g/L，MgSO 4 0.1-1 g/L, water as solvent and pH7.0.
4. The use according to claim 3, wherein the fermentation medium has a final concentration composition of: 55g/L of starch, 45g/L of dextrin, 10g/L of glucose, 15g/L of yeast powder, 3g/L of peptone, (NH) 4 ) 2 SO 4 0.7g/L，CaCO 3 8g/L，MgSO 4 0.8g/L，K 2 HPO 4 0.8g/L, solvent is water, pH7.0.
5. The use according to claim 3, wherein the fermentation conditions are 28 ℃ and 200rpm fermentation culture for 180h.
6. The application of claim 3, wherein before fermentation, the Streptomyces tsukubaensis ZJXM0001 is inoculated into a seed culture medium, and a seed solution obtained by culture is inoculated into the fermentation culture medium for fermentation culture according to the inoculum concentration of 5-15% by volume, and the seed culture process is as follows: inoculating Streptomyces tsukubaensis ZJXM0001 to a GYM solid culture medium, and culturing at 28 deg.C for 4-7 days; selecting a single colony, inoculating the single colony into a seed culture medium, and culturing at 28 ℃ and 220rpm for 48 hours to obtain a seed solution; composition of GyM solid medium: 10g/L of malt extract, 4g/L of glucose, 4g/L of yeast extract, 20g/L of agar powder, 2g/L of calcium carbonate and water as solvent, wherein the pH value is 7.0; the seed culture medium comprises the following components: 20-35 g/L of glycerin, 5-15 g/L of starch, 5-15 g/L of glucose, 5-15 g/L of yeast powder and CaCO 3 0.5-1.5 g/L, naCl 5-15 g/L, water as solvent and pH7.
CN202210913412.9A 2022-07-25 2022-07-25 Streptomyces tsukubaensis ZJXM0001 with high tacrolimus yield and low ascomycin yield and application thereof Pending CN115386511A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109735467A (en) * 2019-01-30 2019-05-10 福建省微生物研究所 A kind of streptomycete mutagenic strain of high yield tacrolimus and its application
CN112111482A (en) * 2020-08-31 2020-12-22 浙江工业大学 Method and strain for screening high-yield tacrolimus Streptomyces tsukubaensis by high-throughput mutagenesis
CN112852635A (en) * 2021-01-18 2021-05-28 浙江工业大学 Tacrolimus-producing strain capable of rapidly growing and application thereof
CN113717892A (en) * 2021-09-13 2021-11-30 福建省微生物研究所 Streptomyces tsukubaensis strain for producing tacrolimus through fermentation and application thereof
CN113980873A (en) * 2021-12-09 2022-01-28 浙江工业大学 Streptomyces tsukubaensis SCLX0001 and application thereof in fermentation production of tacrolimus

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109735467A (en) * 2019-01-30 2019-05-10 福建省微生物研究所 A kind of streptomycete mutagenic strain of high yield tacrolimus and its application
CN112111482A (en) * 2020-08-31 2020-12-22 浙江工业大学 Method and strain for screening high-yield tacrolimus Streptomyces tsukubaensis by high-throughput mutagenesis
CN112852635A (en) * 2021-01-18 2021-05-28 浙江工业大学 Tacrolimus-producing strain capable of rapidly growing and application thereof
CN113717892A (en) * 2021-09-13 2021-11-30 福建省微生物研究所 Streptomyces tsukubaensis strain for producing tacrolimus through fermentation and application thereof
CN113980873A (en) * 2021-12-09 2022-01-28 浙江工业大学 Streptomyces tsukubaensis SCLX0001 and application thereof in fermentation production of tacrolimus

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