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CN107201316B - Aspergillus and producing pneumocandin B thereof0Method (2) - Google Patents

Aspergillus and producing pneumocandin B thereof0Method (2) Download PDF

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CN107201316B
CN107201316B CN201710575306.3A CN201710575306A CN107201316B CN 107201316 B CN107201316 B CN 107201316B CN 201710575306 A CN201710575306 A CN 201710575306A CN 107201316 B CN107201316 B CN 107201316B
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李娜
江沛
郑玲辉
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Zhejiang Hisun Pharmaceutical Co Ltd
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Abstract

The invention discloses a novel aspergillus strain and application thereof. The strain is named as Glarea lozoyensis HS-2158, and the preservation number is CGMCC NO. 13367. The strain HS-2158 of the invention is pneumocandin B0High yield bacteria and produced pneumocandin C0、E0、B5、B0Serine analogue and other by-products accounting for pneumocandin B0The percentage content of the compound is lower, and the pneumocandin B is reduced0The extraction and separation difficulty of the strain HS-2158 is high, so that the strain HS-2158 has a high application value.

Description

Aspergillus and producing pneumocandin B thereof0Method (2)
Technical Field
The invention relates to the technical field of microbial engineering, in particular to aspergillus and application thereof in preparation of pneumocandin B0(Pneumocandin B0) The use of (1).
Background
Caspofungin (Caspofungin) was developed by merck corporation under the trade name of science (cantidae), and has a structural formula shown in formula 1 below, was first marketed in the united states in 2001, and has been widely used in more than 70 countries. At present, the product becomes a king brand drug in the market of antifungal drugs for the whole body.
Figure BDA0001350792760000011
Caspofungin is a glucan synthase inhibitor (which is not found in mammalian cells and is required for many pathogenic fungi to synthesize a major part of the cell wall.) it catalyzes the transport of the glucan group in uridine diphosphate to β -1, 3-D-glucan β -1, 3-D-glucan synthase, which is essential for fungal growth, inhibiting the enzyme to disrupt cell wall structure and leak cell contents. caspofungin has broad spectrum antifungal activity, and can treat fluconazole-resistant candida, aspergillus, and certain endemic mycoses, and can be effective against pneumocystis, etc. by virtue of its activity in β -1, 3-D-glucan synthase in non-competitively inhibited fungal cell walls, which in turn causes the lysis of fungal cell walls and the change in intracellular and extracellular osmotic pressure, thereby killing fungal cells completely.
Pulmonary monisin B0(Pneumocandin B0) Is a raw material for synthesizing caspofungin, is a natural antifungal lipopeptide antibiotic produced by Glarea zoyensis, and the Glarea zoyensis can produce A0And B0In addition to the two main products, C can also be produced0、D0、E0、B1And B2Waiting for 16 analogs, different amino acid side chain modifications or different amino acid linkage sequences lead to structural diversity of pneumocandin compounds. The structures of currently known pneumocandin compounds and analogues thereof are shown in the following formula 2 and the following table 1:
Figure BDA0001350792760000021
TABLE 1 substituents of pneumocandin compounds
Pneumocandin R1 R2 R3 R4 R5 R6 R7
A0 CH3 OH OH OH CH3 OH OH
B0 H OH OH OH CH3 OH OH
C0 OH H OH OH CH3 OH OH
D0 OH OH OH OH CH3 OH OH
E0 H H OH OH CH3 OH OH
A1 CH3 OH OH OH CH3 H OH
A2 CH3 OH H H CH3 OH OH
A3 CH3 OH OH H CH3 H H
A4 CH3 OH H H CH3 H H
B1 H OH OH OH CH3 H OH
B2 H oH H H CH3 OH OH
B5 H OH OH H CH3 OH OH
B6 H oH H oH CH3 OH OH
D2 OH OH H H CH3 OH OH
B0Serine analogue H OH OH OH H OH OH
B5Serine analogue H OH OH H H OH OH
Qin TingEt al in the antibiotic journal of China, 2016, volume 41, volume 3 (protoplast mutagenesis screening of high-yielding pneumocandin B0Strain of (d) reported that pulmonary candidin B was selectively developed by nitrosoguanidine mutagenesis by Merck0The source of the producing strain ATCC74030, the fermentation titer is 241 mu g/ml, and the pneumocandin B is obtained under the condition that mannitol is used as a carbon source through carbon source optimization0The titer reaches 800 mug/ml. Then the preparation and regeneration of protoplast are carried out, and the pneumocandin B is screened out by adopting normal pressure room temperature plasma (ARTP) mutagenesis0The high-yield strain has the titer of 1130 mu g/ml.
WO00/08197 discloses the fermentative preparation of pneumocandin B using strain ATCC740300In the method, 125g/L of fructose is used as a carbon source, and amino acid and trace elements are added to reduce impurities, wherein 15g/L of proline is added to reduce pneumocandin C0Reduced to 4.4% but pneumocandin E0Increasing to 2.7%, adding zinc to make pneumocandin B0Serine analogs and B5The content is respectively reduced by 1%, but pneumocandin E0Has one-time increased content and pneumocandin B0The titer is reduced by 50%, and pneumocandin E can be obtained by adding trace element cobalt0The content was reduced from 2.2% to 1.8%, but pneumocandin B0Serine analogs and B5Doubling the content and pneumocandin B0The titer is reduced by 25 percent, and amino acid and trace elements are added in the fermentation process, thereby increasing the production cost.
Can effectively separate pneumocandin B even through a large-volume silica gel column0Structural analogs of (4), but increasing pneumocandin B at the bacterial level0The titer of (A) and the simultaneous reduction of the content of by-products, thereby reducing the cost of downstream separation, is necessary, and therefore, a new, highly effective, low by-product, easily extractable pneumocandin B is sought0(Pneumocandin B0) The work of producing the strain of (1) is still ongoing.
Disclosure of Invention
One of the purposes of the invention is to provide a novel strain Glarea lozoyensis HS-2158 with the preservation number of CGMCC NO. 13367.
The invention also aims to provide the strain HS-2158 for preparing pneumocandin B0Or an analogue thereof, said analogue being pneumocandin A0，C0，D0，E0，A1，A2，A3，A4，B1，B2，B5，B6，D2，B0Serine analogues, B5Serine analogs, preferably pneumocandin C0、B5、E0、B0A serine analog.
The invention also provides a method for preparing pneumocandin B by adopting the strain HS-21580The method of (1). The method comprises the steps of carrying out colony culture on the strain HS-2158 in a solid culture medium, then carrying out seed culture in a seed culture medium, and then inoculating in a fermentation culture medium for fermentation culture.
In a preferred embodiment, the colony is cultured for 5 to 15 days, preferably 7 to 12 days; the time for seed culture is 24-120 hours, preferably 48-100 hours; the temperature of the seed culture is 20-30 ℃, and preferably 22-28 ℃. The time of the fermentation culture is 96-360 hours, preferably 216-340 hours; the temperature of the fermentation culture is 20-30 ℃, and preferably 22-28 ℃.
In a preferred embodiment, the concentration of each component of the seed culture medium in the culture medium is: 5.0-70.0 g/L of carbon source, 5.0-60.0 g/L of nitrogen source, 0-5.5 g/L of inorganic salt, and the pH value of the seed culture medium is 5.0-7.0; and/or
The concentration of each component of the fermentation medium in the culture medium is as follows: 5.0-170.0 g/L of carbon source, 5.0-65.0 g/L of nitrogen source, 0-21.0 g/L of inorganic salt and 5.0-30.0 g/L of amino acid, wherein the pH value of the fermentation medium is 5.0-7.5;
in a preferred embodiment, the carbon source of the seed culture medium is selected from one or more of glucose, corn starch, lactose and maltodextrin, the nitrogen source of the seed culture medium is selected from one or more of soybean cake powder, cottonseed cake powder, peptone, peanut cake powder and corn steep liquor dry powder, and the inorganic salt of the seed culture medium is selected from one or more of sodium nitrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium nitrate, ammonium sulfate, calcium carbonate and zinc sulfate; and/or
The carbon source of the fermentation medium is selected from one or more of glucose, lactose, fructose, mannitol, sorbitol, maltodextrin, corn starch, soybean oil and methyl oleate, the nitrogen source of the fermentation medium is selected from one or more of yeast extract powder, yeast powder, beef extract, soybean peptone, soybean cake powder, cottonseed cake powder, peanut cake powder, gluten powder, corn steep liquor dry powder and bran, the inorganic salt of the fermentation medium is selected from one or more of ammonium chloride, ammonium nitrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, calcium carbonate, ferrous sulfate, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, ferric chloride, manganese sulfate, zinc sulfate and copper sulfate, and the amino acid of the fermentation medium is selected from one or more of L-tyrosine, L-proline, L-threonine, L-ornithine hydrochloride, One or more of L-valine and L-arginine.
Preferably, the seed culture medium contains 5.0-20.0 g/L of corn starch, 10.0-50.0 g/L of glucose, 10.0-30.0 g/L of soybean cake powder, 5.0-15.0 g/L of peptone, 5.0-15.0 g/L of peanut cake powder, 0.5-2.0 g/L of potassium dihydrogen phosphate, 0.5-1.5 g/L of ammonium sulfate and 0.5-2.0 g/L of calcium carbonate, and the pH value of the seed culture medium is 6.0-7.0; and/or
The fermentation medium contains 90.0-150.0 g/L of sorbitol, 5.0-50.0 g/L of glucose, 5.0-20.0 g/L of soybean cake powder, 5.0-15.0 g/L of peptone, 10.0-30.0 g/L of cottonseed cake powder, 1.0-7.0 g/L of monopotassium phosphate, 0.5-4.0 g/L of ammonium sulfate, 0.5-5.0 g/L of calcium chloride, 0.8-5.0 g/L, L-5.0 g/L of zinc sulfate, and the pH value of the fermentation medium is 5.0-7.0.
More preferably, the seed culture medium contains 10.0g/L of corn starch, 30.0g/L of glucose, 20.0g/L of soybean cake powder, 8.0g/L of peptone, 10.0g/L of peanut cake powder, 1.0g/L of potassium dihydrogen phosphate, 0.8g/L of ammonium sulfate and 1.0g/L of calcium carbonate, and the pH value of the seed culture medium is 6.5; and/or
The fermentation medium contains 100.0g/L sorbitol, 5.0g/L glucose, 20.0g/L soybean cake powder, 5.0g/L peptone, 30.0g/L cottonseed cake powder, 5.0g/L potassium dihydrogen phosphate, 3.0g/L ammonium sulfate, 4.0g/L calcium chloride and 1.0g/L, L-proline 15.0g/L zinc sulfate, and the pH of the fermentation medium is 7.0.
Pulmonary monisin B0And analogs thereof can be detected by liquid phase (HPLC) under the following conditions:
a. pulmonary monisin B0、B5、E0、B0High performance liquid phase analysis method of serine analogue: a chromatographic column: ODS column 4.6 mm. times.250 mm, column temperature: 40 ℃; mobile phase acetonitrile water 60: 40 (volume ratio), flow rate: 1.0ml/min, detection wavelength: 210nm, sample size: 10 mu l of the mixture;
b. pulmonary monisin B0And C0The high performance liquid phase analysis method of (1): a chromatographic column: the silicon column is 4.6mm multiplied by 250 mm; mobile phase, n-hexane, methanol and water (volume ratio) 70: 30: 2, flow rate: 1.0ml/min, detection wavelength: 276nm, sample size: 10 μ l.
Remarking: when HPLC detection is carried out, firstly, the pneumocandin B is detected by a detection method a0、B5、E0、B0The titer of the serine analogue is then detected by the detection method B to obtain pneumocandin B0And C0In the detection method a, and then detecting pneumocandin B0Titer of pneumocandin C0The potency of (A).
The viscosity of the invention can be detected under the following conditions, and the instrument comprises: brokfield, rotor: 16s, rotation speed: 30 rpm.
The strain HS-2158 of the invention and pneumocandin B in the prior art0Compared with the producing strain, the producing strain has the following advantages:
① production of pneumocandin B by the strain HS-2158 of the invention0Has high fermentation unit and can stably produce pneumocandin B0。
② the strain HS-2158 of the present invention reduces pneumocandin C0、E0、B5、B0Serine analogue and other by-products accounting for pneumocandin B0In a percentage content of pulmonary monisin B0Difficulty in extraction and separation of (A) fromIs favorable for obtaining high-purity pneumocandin B0。
③ Prior Art discloses pneumocandin B0The fermentation carbon source of the producing strain is fructose or mannitol, the strain HS-2158 can well utilize sorbitol, can be fermented in a fermentation medium taking sorbitol as a main carbon source, and the pneumocandin B0The maximum titer of the fermentation broth can reach 7625 mug/ml, which is far higher than other known production bacteria, the viscosity of the fermentation broth obtained after the culture is low, and the purification difficulty of the fermentation product is reduced.
④ Prior Art discloses that by adding proline, threonine, trace elements zinc, nickel, etc. to reduce some impurities respectively, the strain HS-2158 of the present invention can reduce pneumocandin C at the same time0、E0、B5、B0Serine analogue and other by-products accounting for pneumocandin B0The percentage content of (A) is as follows.
⑤ the strain HS-2158 of the invention has good genetic stability and good industrial application prospect.
Pneumocandin B used in the invention0The producing strain is Glarea lozoyensis HS-2158(CGMCC NO. 13367).
The microbial strain of the strain HS-2158(CGMCC NO.13367) is preserved in the China general microbiological culture Collection center (the address: West Lu No.1 of Beijing Korean district, No. 3 of Xilu, Beijing, institute of microbiology, China academy of sciences) on 09.01.2017, the preservation number is CGMCC NO.13367, the strain is classified and named as Glarea lozoyensis, and the strain is registered in a book to prove survival.
Detailed Description
The reagents used in the following examples to adjust the pH of the media feed are NaOH solutions or hydrochloric acid, which are conventional in the art.
In the following examples, the solid medium was made to a constant volume with deionized water, the seeds and the fermentation medium were made to a constant volume with tap water, the raw materials were weighed according to the formulation for each medium, the raw materials were mixed thoroughly, water was added to the required volume, and finally the pH was adjusted to the required value with NaOH solution or hydrochloric acid.
EXAMPLE 1 Strain origin
The strain HS-2158 is an original strain separated from Xinanjiang Zhejiang and a mutant strain obtained by mutagenesis.
After culturing the original strain in PDA slant culture medium at 25 deg.C for 13 days, scraping off mycelium under aseptic condition with inoculating shovel, grinding mycelium in ground test tube and suspending in sterile water to obtain bacterial suspension for NTG (nitrosoguanidine) mutagenesis treatment.
Weighing NTG crystal 5mg, dissolving in 5ml sterile Tris buffer (pH8.0), sucking 3ml NTG solution with pipette, adding into 2ml bacterial suspension, and placing in 25 deg.C culture medium on rotary or reciprocating shaking table for 30 min. The remaining specific steps are as follows:
(1) preparation and culture of mycelia
Sterilizing PDA solid culture medium at 121 deg.C for 30min, cooling to 50-60 deg.C, pouring into plate, diluting the treated bacterial suspension, sucking 0.1ml of bacterial suspension, coating on PDA plate, diluting the bacterial suspension without mutagenesis, coating on PDA plate as control, culturing in constant-temperature incubator at 25 deg.C, culturing bacterial colony for 13 days, and collecting mature mycelia.
(2) Preparation and culture of seed liquid
Seed culture medium formula (g/L): 10.0 parts of corn starch, 30.0 parts of glucose, 20.0 parts of soybean cake powder, 8.0 parts of peptone, 10.0 parts of peanut cake powder, 1.0 part of monopotassium phosphate, 0.8 part of ammonium sulfate and 1.0 part of calcium carbonate, wherein the pH value is 6.5, the liquid filling amount in a shake flask is 20ml/250ml, and the shake flask is sterilized for 30 minutes at 121 ℃. About 0.5 colony is inoculated to each seed shake flask, the culture temperature is 22-28 ℃, the culture humidity is 40-60%, the amplitude of the shake flask machine is 5cm, the rotation speed is 250rpm, and the culture period is 88 hours.
(3) Pulmonary monisin B0Preparation and culture of fermentation medium
Fermentation medium formula I (g/L): 100.0 parts of maltodextrin, 5.0 parts of glucose, 20.0 parts of soybean cake powder, 5.0 parts of peptone, 30.0 parts of cottonseed cake powder, 5.0 parts of monopotassium phosphate, 3.0 parts of ammonium sulfate, 4.0 parts of calcium chloride, 1.0 part of zinc sulfate and 15.0 parts of L-proline; pH7.0, the liquid loading in the shake flask is 30ml/250ml, and the flask is sterilized at 121 ℃ for 30 minutes. Inoculating the seed solution into a fermentation culture medium with the inoculation amount of 10% (volume percentage), wherein the culture temperature is 22-28 ℃, the culture humidity is 40-60%, the amplitude of a bottle shaking machine is 5cm, the rotation speed is 250rpm, and the culture period is 239 hours.
Selecting 1000 strains of single colony after NTG mutagenesis, performing shake flask fermentation, and detecting pneumocandin B by HPLC0The yield of (2). Screening for productive pneumocandin B0The highest producing mutant strain was strain HS-2158. The main biological characteristics are as follows: the mature bacterial colony is black green, produces yellow green spores, is tall and solid, has a slightly irregular round shape and a diameter of 0.8-1.2cm, and does not produce water-soluble pigment.
Example 2 species identification
Experiments are carried out according to the relevant contents in books such as general mycology, a handbook of identifying common bacteria systems, a guide for molecular cloning experiments, Chinese pharmacopoeia (2005 edition XIH) and the like.
1. The culture characteristics are as follows: the color and pigment condition of the mycelium are observed after 10 culture media of ISP1, ISP2, ISP3, ISP4, ISP5, Chachi, PDA, calcium malate and Gao's I culture medium are cultured for 7-10 days at 28 ℃. The culture characteristics of the strains are shown in Table 3.
TABLE 3 culture characteristics of the strain HS-2158(CGMCC NO.13367) on 10 media
Culture medium Growth conditions Substrate hypha Aerial hypha Soluble pigments
ISP1 2 Olive green White colour Is free of
ISP2 4 Olive green Yellow green Is free of
ISP3 4 Olive green Yellow green Is free of
ISP4 1 Yellow green White colour Is free of
ISP5 1 Light pink color Light pink color Is free of
Chashi 2 Brown green White colour Is free of
PDA 4 Olive green Yellow green Is free of
Calcium malate 3 Black green White colour Is free of
Nutrition 3 Brown black color Brown colour Is free of
Gao's number one 1 Brown green White colour Is free of
2. Physiological and biochemical tests: except for the temperature experiment, the culture is carried out for 5-7 days at 28 ℃.
a) Utilization of carbon source: using ISP9 as the basal medium, the final concentration of each carbon source was 1.0%, as shown in Table 4.
b) Utilization of inorganic nitrogen source: using ISP9 as the basal medium, the potassium nitrate and ammonium sulfate concentrations were 1.0% each, as shown in Table 4.
c) The basic culture medium adopted in the degradation test and the NaCl tolerance test is GYEA (pH6.0), the results of the degradation test are shown in Table 5, and the results of the NaCl tolerance test are shown in Table 6.
d) The oxidase and catalase tests, the pH tests and the temperature tests all adopt PDA culture medium. The oxidase and catalase test results are shown in Table 7, the pH test results are shown in Table 8, and the temperature test results are shown in Table 9.
e) M.R, V.P experiments: the method of 'Manual of identification of common bacteria system' is adopted. The results are shown in Table 7.
Physiological and biochemical characteristics: see tables 4-9.
TABLE 4 utilization of carbon and nitrogen sources by the strain HS-2158(CGMCC NO.13367)
Figure BDA0001350792760000071
Figure BDA0001350792760000081
TABLE 5 degradation test results of the strain HS-2158(CGMCC NO.13367)
Degradation product Concentration of degradants Results ＊ Degradation product Concentration of degradants Results
Adenine 0.5％ 3，- Casein protein 1.0％ 4，-
Guanine and its preparing process 0.5％ 3，- Tyrosine 1.0％ 3，-
Xanthine 0.4％ 3，- Tween-40 1.0％ 4，-
Hypoxanthine 0.4％ 2，- Tween-60 1.0％ 3，-
Xylan 0.4％ 3，- Tween-80 1.0％ 2，-
TABLE 6 tolerance of strain HS-2158(CGMCC NO.13367) to NaCl
Concentration of NaCl 1％ 4％ 7％ 10％
Growth of the Strain 3 1 0 0
TABLE 7 Main physiological and biochemical characteristics of the strain HS-2158(CGMCC NO.13367)
Test items Results Test items Results Test items Results
Liquefaction of gelatin + Hydrogen sulfide generation - Catalase enzyme -
Starch hydrolysis - V.P experiment - Oxidase enzyme -
Milk coagulation - M.R experiment -
Milk peptone - Nitrate reduction +
TABLE 8 pH test for the growth of the Strain HS-2158(CGMCC NO.13367)
pH 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5
Growth conditions 3 3 3 3 4 4 4 4 4 4
TABLE 9 temperature test for growth of Strain HS-2158(CGMCC NO.13367)
Temperature (. degree.C.) 7 14 28 37 45
Growth conditions 0 2 4 3 1
Injecting: in tables 3 to 9: 0, no growth; 1, very weak growth; 2, the fertilizer can grow and has a small amount of spores; 3, the growth is good, and a large number of spores exist; 4, the growth is best, and spores are abundant; positive; negative.
3.26S rDNA sequence analysis: collecting fresh thallus cultured by PDA, extracting total DNA template by liquid nitrogen wall breaking method, carrying out 26S rDNA gene D1/D2 region amplification by using Fungi Identification PCR Kit (TaKaRa), directly carrying out sequence determination after agarose electrophoresis detection of PCR product, and purifying and sequencing the PCR product by Shanghai biological engineering technology company Limited. The 26S rDNA gene D1/D2 region sequence measured by strain HS-2158(CGMCC NO.13367) is collated and then compared with the homologous sequence BLAST of related species and genus in GenBank database to determine the classification status of the strain.
The 26S rDNA D1/D2 sequence (see sequence Listing) of HS-2158(CGMCC NO.13367) was BLAST-compared with the related sequences in GenBank, and the results are shown in Table 10 (only the strains with higher homology are listed in the Table).
TABLE 10 homology of the strain HS-2158(CGMCC NO.13367) and related strains
Figure BDA0001350792760000091
The strain HS-2158(CGMCC NO.13367)26S rDNA D1/D2 is subjected to region sequencing, has high homology with aspergillus (Glarea lozoyensis) and the highest homology reaches 99%, and meanwhile, the strain HS-2158(CGMCC NO.13367) is subjected to apparent characteristic test, and is found to be very close to related classification parameters of the aspergillus (Glarea lozoyensis), so that the strain HS-2158(CGMCC NO.13367) is identified as the strain of the aspergillus (Glarea lozoyensis).
Example 3 Strain HS-2158(CGMCC NO.13367) when the main carbon sources of the fermentation Medium were sorbitol and maltodextrin
Study of fermentation Process
(1) Sterilizing PDA solid culture medium at 121 deg.C for 30min, cooling to 50-60 deg.C, pouring into flat plate, inoculating 0.1ml bacterial suspension, culturing for 13d, and collecting mature mycelia.
(2) The formulation of the seed medium and the culture conditions were the same as those in step (2) of example 1, and the culture period was 87 hours.
(3) Pulmonary monisin B0Preparing and culturing a fermentation medium.
Fermentation medium formula I was identical to step (3) in example 1;
fermentation medium formula II (g/L): 50.0 of sorbitol, 50.0 of maltodextrin, 5.0 of glucose, 20.0 of soybean cake powder, 5.0 of peptone, 30.0 of cottonseed cake powder, 5.0 of monopotassium phosphate, 3.0 of ammonium sulfate, 4.0 of calcium chloride, 1.0g of zinc sulfate, 15.0 of L-proline and pH 7.0;
fermentation medium formula III (g/L): 100.0 parts of sorbitol, 5.0 parts of glucose, 20.0 parts of soybean cake powder, 5.0 parts of peptone, 30.0 parts of cottonseed cake powder, 5.0 parts of monopotassium phosphate, 3.0 parts of ammonium sulfate, 4.0 parts of calcium chloride, 1.0 part of zinc sulfate, 15.0 parts of L-proline and 7.0 parts of pH.
The liquid loading in the shake flask is 30ml/250ml, and the shake flask is sterilized for 30 minutes at 121 ℃. Inoculating the seed solution into a fermentation culture medium with the inoculation amount of 10% (volume percentage), wherein the culture temperature is 22-28 ℃, the culture humidity is 40-60%, the amplitude of a bottle shaking machine is 5cm, the rotation speed is 250rpm, and the culture period is 240 hours. The results of the three fermentations are shown in Table 11.
TABLE 11 fermentation results of HS-2158(CGMCC NO.13367) in different fermentation formulations
Figure BDA0001350792760000092
Figure BDA0001350792760000101
FM I: fermentation medium formula I
FM II: fermentation medium formula II
FM III: fermentation medium formula III
As can be seen from the table, when the carbon source of the fermentation medium is maltodextrin or sorbitol, the strain HS-2158 produces pneumocandin B0The titer of the product is higher, wherein sorbitol is preferred as a carbon source, the fermentation titer is higher, and the viscosity is lower.
Example 4 fermentation Process study of Strain HS-2158(CGMCC NO.13367) when carbon source in fermentation Medium is maltodextrin and sorbitol
(1) Sterilizing PDA solid culture medium at 121 deg.C for 30min, cooling to 50-60 deg.C, pouring into flat plate, inoculating 0.1ml bacterial suspension, culturing for 13d, and collecting mature mycelia.
(2) The formulation of the seed medium and the culture conditions were the same as those in step (2) of example 1, and the culture period was 87 hours.
(3) Pulmonary monisin B0Preparing and culturing a fermentation medium.
Fermentation medium formula I was identical to step (3) in example 1;
fermentation medium formula III (g/L): 100.0 parts of sorbitol, 5.0 parts of glucose, 20.0 parts of soybean cake powder, 5.0 parts of peptone, 30.0 parts of cottonseed cake powder, 5.0 parts of monopotassium phosphate, 3.0 parts of ammonium sulfate, 4.0 parts of calcium chloride, 1.0 part of zinc sulfate, 15.0 parts of L-proline and 7.0 parts of pH.
The liquid loading in the shake flask is 30ml/250ml, and the shake flask is sterilized for 30 minutes at 121 ℃. Inoculating the seed solution into a fermentation culture medium with the inoculation amount of 10% (volume percentage), wherein the culture temperature is 22-28 ℃, the culture humidity is 40-60%, the amplitude of a bottle shaking machine is 5cm, the rotation speed is 250rpm, and the culture period is 240 hours. The results of the three fermentations are shown in Table 12, as measured by viscosity and HPLC.
TABLE 12 comparison of the fermentations of HS-2158(CGMCC NO.13367) on FM I and FM III
Figure BDA0001350792760000102
FM I: fermentation medium formula I
FM III: fermentation medium formula III
As can be seen from the table, whether the carbon source of the fermentation medium is sorbitol or maltodextrin, pneumocandin C produced by the strain HS-2158 of the present invention0、E0、B5、B0Serine analogue and other by-products accounting for pneumocandin B0Are close and all lower.
In comparison with the published patent WO00/08197, this patent specifically discloses that pneumocandin C can be converted by addition of 15g/L proline0Reduced to 4.4% but pneumocandin E0Increasing to 2.7%, adding zinc to make pneumocandin B0Serine analogs and B5The content is respectively reduced by 1 percent (B)0Serine analogue 1.9%, B53.9%) but pneumocandin E0The content is increased by one time (pneumocandin E)04.3%) and pneumocandin B0The titer is reduced by 50%, and pneumocandin E can be obtained by adding trace element cobalt0The content was reduced from 2.2% to 1.8%, but pneumocandin B0Serine analogs and B5The content is doubled (pneumocandin B)0Serine analogue 6.0%, B57.2%) and pneumocandin B0The titer is reduced by 25%.
Comparison revealed that the fermentation contains proline, but pneumocandin C of the present patent0The percentage of (A) is lower; pulmonary monisin E0、B5、B0Compared with the published patent process, the percentage content of the byproducts such as serine analogs is lower; in addition, the patent has disclosed that certain impurities are reduced by adding proline, trace elements such as zinc and nickel, respectively, and the strain HS-2158 of the invention canSimultaneous reduction of pneumocandin C0、E0、B5、B0Serine analogue and other by-products accounting for pneumocandin B0The percentage content of (A) is as follows.
Example 5 fermentation Process study of Strain HS-2158(CGMCC NO.13367) with different concentrations of sorbitol as carbon Source in fermentation Medium
(1) Solid Medium formulation and culture conditions were the same as those in the step (1) of example 3
(2) The formulation of the seed medium and the culture conditions were the same as those in step (2) of example 1, and the culture period was 87 hours.
(3) Pulmonary monisin B0Preparation and culture of fermentation medium
A basic formula IV (g/L) of a fermentation culture medium: 5.0 parts of glucose, 20.0 parts of soybean cake powder, 5.0 parts of peptone, 30.0 parts of cottonseed cake powder, 5.0 parts of monopotassium phosphate, 3.0 parts of ammonium sulfate, 4.0 parts of calcium chloride, 1.0 part of zinc sulfate and 15.0 parts of L-proline. Sorbitol is added into the basic formula IV of the fermentation culture medium, the concentration of the sorbitol is respectively 90.0g/L, 110.0g/L, 130.0g/L and 150.0g/L, four groups of fermentation culture mediums with different concentrations of sorbitol are obtained, the pH value is 7.0, the liquid filling amount of a shake flask is 30ml/250ml, and the four groups of fermentation culture mediums are sterilized for 30 minutes at the temperature of 121 ℃. Inoculating the seed solution into the four groups of fermentation culture media with sorbitol of different concentrations in an inoculation amount of 10 percent (volume percentage), wherein the culture temperature is 22-28 ℃, the culture humidity is 40-60 percent, the amplitude of a bottle shaking machine is 5cm, the rotation speed is 250rpm, and the culture periods are 240 hours and 286 hours respectively. HPLC analysis, the results are shown in Table 12.
TABLE 13 comparison of sorbitol fermentation results at different concentrations
Sorbitol concentration (g/L) 90.0 110.0 130.0 150.0
240 hours HPLC B0(μg/ml) 5613 6711 6102 5615
286 h HPLC B0(μg/ml) 4522 7194 7625 7013
EXAMPLE 6 bench study of the Strain HS-2158(CGMCC NO.13367) on a 50L fermenter
(1) Preparation of seed liquid in seed tank
Putting 10L of seed culture medium (the proportion of the seed culture medium is the same as that in example 1, 0.05 percent of PPG is added as an antifoaming agent) into a 15L seeding tank, performing steam sterilization for sterilization, performing 30 minutes at 121 ℃, cooling, then adding 200ml of seed solution in a shake flask, performing culture at 22-28 ℃, stirring at 100-500 rpm, ventilating at 0.5-1.0 vvm, and dissolving oxygen at 20-70%, and performing culture for 86 hours.
(2) Preparation and culture of fermentation tank culture medium
The formula of the fermentation medium is the same as that of the fermentation formula III in the previous example 3, but 0.05 percent PPG is added as an antifoaming agent, the filling amount of a fermentation tank is 35L/50L, the pH value is 7.0, steam sterilization is carried out for 30 minutes at 121 ℃, about 3.5L of seed tank culture solution is inoculated after cooling, the fermentation temperature is 22-28 ℃, the stirring speed is 200-500 rpm, the ventilation amount is 0.5-1.0 vvm, the fermentation culture is 252 hours, HPLC detection is carried out on the fermentation liquid during the tank discharge of the fermentation tank, and then pneumocandin B is measured0The fermentation titer was 7010. mu.g/ml. C0Efficiency of fermentationA valence of 156. mu.g/ml, B5Fermentation titer 44. mu.g/ml, E0Fermentation titer 32. mu.g/ml, B0The fermentation titer of the serine analogue is 34 mu g/ml.
Example 7 amplification culture of the Strain HS-2158(CGMCC NO.13367) on a 5000L fermenter
(1) Preparation of seed liquid in seed tank
300L of seed culture medium (the proportion of the seed culture medium is the same as that in example 1, 0.05 percent of PPG is added as an antifoaming agent) is put into a 500L seeding tank, steam sterilization is adopted for sterilization, 30 minutes are carried out at 121 ℃, 1800ml of seed solution in a shake flask is added after cooling, the culture temperature is 22-28 ℃, the stirring speed is 50-200 rpm, the ventilation volume is 0.6-1.2 vvm, the dissolved oxygen is 30-70 percent, and the seed solution is cultured for 98 hours.
(2) Preparation and culture of fermentation tank culture medium
The formula of the fermentation medium is the same as that of the fermentation formula III in the previous example 3, but 0.05 percent PPG is added as a defoaming agent, the filling amount of a fermentation tank is 3000L/5000L, the pH value is 7.0, steam sterilization is carried out for 30 minutes at 121 ℃, after cooling, about 300L of seeding tank culture solution is inoculated, the fermentation temperature is 22-28 ℃, the stirring rotation speed is 300-500 rpm, the ventilation amount is 0.5-1.5 vvm, the fermentation culture is 280 hours, the fermentation liquid is subjected to HPLC detection when the fermentation tank is placed, and the pneumocandin B is measured0The fermentation titer was 6845. mu.g/ml. C0Fermentation titer 138. mu.g/ml, B5Fermentation titer 38. mu.g/ml, E0Fermentation titer 34. mu.g/ml, B0The fermentation titer of the serine analogue is 29 mug/ml.
Although specific embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are merely examples and that many variations or modifications may be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is therefore defined by the appended claims.
SEQUENCE LISTING
<110> Zhejiang Haizheng pharmaceutical industry Co., Ltd
<120> an aspergillus and a method for producing pneumocandin B0 by using the same
<160>1
<170>PatentIn version 3.3
<210>1
<211>999
<212>DNA
<213> Aspergillus (Glarea lozoyensis)
<400>1
ggatctagac ggcagtgatt ccagctcggt accaccggga tcctctgcga gtgcaccagt 60
aacagctcaa atttgaaatc tggctctttt agggtccgag ttgtaatttg tagaagatgc 120
ttcgggtgta gctccggtct aagttccttg gaacaggacg tcatagaggg tgagaatccc 180
gtatgtgact ggtggctttc gcccatgtga agctctttcg acgagtcgag ttgtttggga 240
atgcagctct aaatgggtgg taaatttcat ctaaagctaa atattggcca gagaccgata 300
gcgcacaagt agagtgatcg aaagatgaaa agcactttgg aaagagagtt aaacagtacg 360
tgaaattgtt gaaagggaag cgcttgcaac cagatttgca ccccgtcgat catcccccgt 420
tctcggaggt gcactcggcg gggttcaggc cagcatcggt ttcggtggtg ggataaaggc 480
cttgggaacg tagctcctct cggggagtgt tatagccctc ggtgcaatgc cgcctaccgg 540
gaccgaggac cgcgcttcgg ctaggatgct ggcgtaatgg ttgtaagcga cccgtcttga 600
aacacggacc cctgtgtgaa attgttatcc gctcaatcgt cgacctgcag gcatgcaagc 660
ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 720
cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 780
ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 840
ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 900
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 960
cactcaaagg cggtaatacg gttatccaca gaatcaggg 999

Claims (8)

1. Pneumocandin B0The method for producing (A), which comprises subjecting a strain toGlarea lozoyensisHS-2158 is cultured in solid culture medium for colony culture, then cultured in seed culture medium for seed culture, and inoculated in fermentation culture medium for fermentation culture; said strainGlarea lozoyensisThe preservation number of HS-2158 is CGMCC NO. 13367; the fermentation medium contains 100.0g/L sorbitol, 5.0g/L glucose, 20.0g/L soybean cake powder, 5.0g/L peptone, 30.0g/L cottonseed cake powder, 5.0g/L potassium dihydrogen phosphate, 3.0g/L ammonium sulfate, 4.0g/L calcium chloride and 1.0g/L, L-proline 15.0g/L zinc sulfate, and the pH of the fermentation medium is 7.0.
2. The method according to claim 1, wherein the colony is cultured for 5 to 15 days.
3. The method according to claim 2, wherein the colony is cultured for 7 to 12 days.
4. The method according to claim 1, wherein the seed culture time is 24 to 120 hours; the temperature of seed culture is 20-30 ℃.
5. The method according to claim 4, wherein the seed culture time is 48 to 100 hours; the temperature of seed culture is 22-28 ℃.
6. The method according to claim 1, wherein the fermentation culture time is 96 to 360 hours; the temperature of the fermentation culture is 20-30 ℃.
7. The method according to claim 6, wherein the fermentation culture time is 216-340 hours; the temperature of the fermentation culture is 22-28 ℃.
8. The method of claim 1, wherein: the seed culture medium contains 10.0g/L of corn starch, 30.0g/L of glucose, 20.0g/L of soybean cake powder, 8.0g/L of peptone, 10.0g/L of peanut cake powder, 1.0g/L of monopotassium phosphate, 0.8g/L of ammonium sulfate and 1.0g/L of calcium carbonate, and the pH value of the seed culture medium is 6.5.
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