WO2012079521A1 - 一种肽类抗生素的高产菌株及其制备方法和用途 - Google Patents

一种肽类抗生素的高产菌株及其制备方法和用途 Download PDF

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WO2012079521A1
WO2012079521A1 PCT/CN2011/084043 CN2011084043W WO2012079521A1 WO 2012079521 A1 WO2012079521 A1 WO 2012079521A1 CN 2011084043 W CN2011084043 W CN 2011084043W WO 2012079521 A1 WO2012079521 A1 WO 2012079521A1
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strain
preparation
formula
fermentation medium
compound
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PCT/CN2011/084043
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English (en)
French (fr)
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陈懿
刘石东
张兆利
王春霞
康静
季晓铭
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上海天伟生物制药有限公司
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Priority to KR1020137018369A priority Critical patent/KR101375421B1/ko
Priority to US13/994,633 priority patent/US8911968B2/en
Priority to CA2821791A priority patent/CA2821791C/en
Priority to AU2011344954A priority patent/AU2011344954B2/en
Priority to EP11849471.5A priority patent/EP2653532B1/en
Priority to RU2013132191/10A priority patent/RU2560258C2/ru
Priority to JP2013543513A priority patent/JP5908923B2/ja
Publication of WO2012079521A1 publication Critical patent/WO2012079521A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to the field of antibiotic preparation, and in particular to a high-yield strain of a peptide antibiotic and a preparation method and use thereof. Background technique
  • echinomycin antibiotics is a group of natural products discovered in the 1970s. It has similar cyclic polypeptide cores and structural features of different fatty acid side chains, which can competitively inhibit ⁇ -D of fungal cell walls. - Synthesis of dextran. The advantages of echinomycins are low toxicity, strong bactericidal activity and excellent pharmacokinetic properties.
  • the echinocandin family includes members such as WF1 1899A, echinocandins, cilostazol, neomoconazoles, echinosporins, and erectin.
  • WF1 1899A members such as WF1 1899A, echinocandins, cilostazol, neomoconazoles, echinosporins, and erectin.
  • Mi cafungi n, echinocandins and pneumocandin are the most in-depth and clinically available antifungal drugs.
  • Mi cafungin is a water-soluble lipopeptide-like echinomycin antifungal drug derived from the chemical modification of the fermented product of P. stipitis CWe ⁇ a ⁇ pe r by Fujisawa Corporation. Developed as a broad spectrum antifungal drug. In an open study conducted in Japan on patients with deep fungal infections (Candida or Aspergillus), the average treatment rate of each dose of this product was 92% after about 22 days. A single-center study in the United States, using 50 to 150 mg of this product with or without other antifungal drugs, for 14 patients with candidaemia, 11 of 12 patients (92 % ) is valid. The lack of hemolytic toxicity and less drug interactions make these drugs even more advantageous than traditional antifungal drugs. 0 ⁇ CH 2 ) 4 CH ;
  • a class of natural antifungal drugs produced by Colephoma empe tri such as Colephoma empe tri, with structure number I, II, III, deposited under the condition of FERM BP-2635, has a low production capacity of only 700 mg/L, industrially produced. The cost is very high.
  • the present invention aims to provide a mutagenized strain of Colephoma empe tri.
  • Another object of the present invention is to provide a process for the preparation of the above mutagenized strain.
  • a further object of the invention is to provide the use of the above mutagenized strains.
  • a mutagenized strain of CW A ⁇ a ⁇ pe r is provided, deposited under the accession number of CGMCC 4129 at the General Microbiology Center of the China Collection of Microorganisms Collections.
  • a method for producing a mutagenized strain provided by the present invention as described above, the method package
  • a process for the preparation of a compound of formula I comprising the steps of: culturing in a fermentation medium at 15-35 ° C as provided by the invention as described above The mutagenized strain gave a compound as shown in formula I.
  • the fermentation medium contains the following components in terms of the total volume of the fermentation medium: corn syrup 5-20 g/l, cottonseed meal 5-30 g/l, yeast extract 6-15 g/l, Starch 10 - 80g / L, glucose 5-20 g / L, inorganic salt 1.5 - 15g / L, trace elements 10 - 50g / L;
  • the inorganic salt is selected from one of the following or a mixture thereof: phosphate, sulfate.
  • the inoculum amount of the mutagenized strain provided by the present invention as described above is 4 to 10 v/v% based on the total volume of the fermentation medium.
  • the present invention provides a stable, high-yielding strain having a higher yield of structural formula I and more in line with industrial production. detailed description
  • the inventors have unexpectedly discovered that the strain Colephoma empe tri FERM BP-2635 is subjected to mutagenesis by nitrosoguanidine (NTG), and after protoplasts are obtained by using lytic enzyme, a highly productive mutant strain can be obtained by screening the protoplasts for regeneration ( Deposit No. CGMCC 4129), the mutant strain is fermented to obtain a high yield of the compound of formula I. On the basis of this, the present invention has been completed.
  • NVG nitrosoguanidine
  • New strain The present invention provides a novel strain for the production of a compound of formula I, which belongs to the class of Colephoma empe tri, which has been deposited at the General Microbiology Center of the China Collection of Microorganisms Collection and Management Committee in Beijing, with the accession number CGMCC 4129, the date of preservation. For August 31, 2010.
  • the present invention provides a method for preparing a novel strain having the accession number CGMCC 4129, which is carried out as follows:
  • the method provided by the present invention includes the steps of:
  • the new strain can be obtained by adding the appropriate amount of NTG to the FERM BP-2635 seed solution (cell dry weight, DCW 5-30 g/l) after 1-3 days of shake flask culture, and continuing to culture. After 1-2 days, the seed solution was centrifuged, washed, and precipitated and suspended, and then the cell was broken by the lytic enzyme (purchased from the Institute of Microbiology, Guangdong province) to obtain protoplasts. The diluted protoplasts were plated on a hypertonic PDA (potato dextrose agar) plate to obtain a recombinant cell single colony. The above single colonies were screened to obtain a mutagenized new strain.
  • the FERM BP-2635 seed solution cell dry weight, DCW 5-30 g/l
  • the seed solution was centrifuged, washed, and precipitated and suspended, and then the cell was broken by the lytic enzyme (purchased from the Institute of Microbiology, Guangdong province) to obtain protoplasts
  • the present invention also provides a method of fermenting a new strain which is induced to obtain a compound of formula I.
  • the method of stimulating fermentation of a new strain and a new strain to obtain a compound of formula I is:
  • the preserved FERM BP-2635 strain glycerol tube was thawed and inoculated into the seed culture medium (loading liquid 50 mL/250 mL), shaken at 25-30 ° C, 200-300 rpm shaker for 3 days, until the dry weight of the hyphae was reached. 5-30g/L or so.
  • the seed medium components are: sucrose 10-20 g/L, yeast extract 4-10 g/L, soy peptone 10-20 g/L, KH 2 P0 4 1.5-2 g/L, MgS0 4 ⁇ 7H 2 0 0.4-1 g/L, trace element 10-50 g/L, initial pH 5.3-6.0. Sterilize at 121 ° C for 20 minutes.
  • Trace elements FeS0 4 ⁇ 7H 2 0 10-20 g/L, MnS0 4 ⁇ H 2 0 10-20 g/L, ZnS0 4 ⁇ 7H 2 0 2-10g/L, CaCl 2 0.7-2.0 g/L, H has 0.56-2.0 g/L, CuCl 2 ⁇ 2H 2 00.25-2.0 g/L, (NH 4 ) 6 Mo 7 0 24 ⁇ 7H 2 0 0.19-2.0 g/L, concentrated hydrochloric acid 500 ml/L.
  • the seed strain of the starting strain is subjected to NTG mutagenesis treatment, and then subjected to lysis of the lytic enzyme, and the resulting protoplast is regenerated to obtain a mutant strain.
  • Protoplasts were applied to hypertonic PDA medium, and single colonies after 10-12 days of culture were inoculated on a slant medium for culture. After 8-10 days, the excavated pieces were inoculated into the seed culture medium (liquid volume 25 mL/250 mL), and cultured at 25-30 ° C, shaking at 280 rpm for 6-10 days. Seed medium inoculated into fermentation medium In a volume of 25 mL/250 mL), incubate at 25- 30 ° C, 200-300 rpm shaker for 6-12 days. After the completion of the culture, the fermentation broth was extracted with methanol, and the content of the compound of the formula I in the fermentation broth was determined by high performance liquid chromatography.
  • composition of each medium involved can be found in the literature Improvement of FR901379 production by mutinous selection and medium optimization, Journal of Bioscience and Bioengineering VOL 107 No. 5, 530 - 534, 2009, Journal of antibiotics, Vol 45, No.12, Dec 1992, 1867-1874.
  • Hypertonic PDA plate medium potato 300 g/L, glucose 20 g/L, agar 15 g/L, sucrose 273.6 g/L, sterilized at 121 °C for 20 minutes.
  • the new strain has good genetic stability and less impurities, it is beneficial to the separation and purification of the products in the production of the compound of formula I, and the process is enlarged, so it is suitable for industrial production.
  • Units in the weight percent by weight in the present invention are well known to those skilled in the art, for example Refers to the weight of the solute in a 100 ml solution.
  • the injection volume is 5 L, and the detection wavelength is 210 nm.
  • Example 1
  • the deposited FERM BP-2635 strain glycerol tube was thawed and inoculated into the seed medium at a 4% inoculation amount (liquid volume 50 mL/250 mL), 25.
  • C. Shake the shaker at 280 rpm for 2 days until the dry weight of mycelium reached 5-30 g/L.
  • the mutagen NTG was added to the seed solution at a concentration of 10 ⁇ g/mL, and the culture was continued for 1 day. After 1 day of culture, 10 mL of the seed solution containing NTG was taken and centrifuged at 5000 rpm for 10 minutes. The resulting precipitate was washed twice with twice the volume of 0.6 M NaCl to remove the medium and NTG.
  • the lethality of the mutagenesis was 85-90%. .
  • Seed medium sucrose 10 g / L, yeast extract 5 g / L, soy peptone 10 g / L, KH 2 P0 4 1.5 g / L, MgS0 4 ⁇ 73 ⁇ 40 0.4 g / L, trace elements 10 g / L, The initial pH was 5.3. Sterilize at 121 ° C for 20 minutes.
  • hyphae 2000 units/mg containing 20mg/mL, 10mg/ml snail Enzyme (5 units/mg) and 10mg/ml cellulase (15 units/mg) enzyme mixture (0.5MNaCl disodium hydrogen phosphate-citrate buffer solution (PH6.0)) 10mL, 30°C Enzymatic hydrolysis at 80 rpm for 5 h.
  • the enzymatically digested reaction solution was filtered with cotton to remove hyphae to obtain a single cell suspension containing only protoplasts.
  • Single colonies after 8 days of culture were selected and inoculated on a slant medium for culture. After 8 days, the colonies of 0. 5-1. Ocm 2 area were inoculated into the seed culture medium (liquid volume 25 mL/250 mL), and a total of 4000 single colonies were cultured at 25 ° C, 280 rpm shaker for 5 days.
  • the seed culture medium was inoculated into the fermentation medium (loading volume 25 mL/250 mL) at a dose of 4%, and cultured at 25 ° C, 280 rpm shaker for 10 days (addition of 5% starch when cultured to 6 days).
  • the fermentation broth was extracted with 50 ml of methanol, and the content of the compound of the formula I in the fermentation broth was determined by high performance liquid chromatography, among which there were 5 high-yielding strains.
  • One of the obtained high-yield strains CGMCC 4129 was rescreened, and the yield of the compound of formula I reached 1. 4 g/L.
  • the new strain CGMCC 4129 obtained in Example 1 was inoculated into the fermentation medium at a seeding rate of 4%, and cultured in a 50 liter fermenter at a culture temperature of 25 ° C, and the pH of the fermentation broth was maintained at 6.5. After 10 days of culture, the yield of the compound of formula I reached 1. 5 g/L (addition of 5% starch when cultured to 6 days).
  • the starting strain and the mutant strain were separately cultured by the culture method of Example 2. After the completion of the culture, the fermentation broth was extracted with twice the volume of methanol, and the content of the compound of the formula I in the fermentation broth was determined by high performance liquid chromatography. Results Table 1
  • composition of each medium involved is as follows:
  • Screening medium potato 300 g/L, glucose 20 g/L, agar 15 g/L, sucrose 273.6 g/L, sterilized at 121 °C for 20 minutes.
  • Incline medium Potato 300 g/L, glucose 20 g/L, agar 15 g/L, sterilized at 121 °C for 20 minutes.
  • Seed medium sucrose 10 g / L, yeast extract 5 g / L, soy peptone 10 g / L, KH 2 P0 4 1.5 g / L, MgS0 4 ⁇ 73 ⁇ 40 0.4 g / L, trace elements 10 g / L, The initial pH was 5.3. Sterilize at 121 ° C for 20 minutes.
  • Fermentation medium corn syrup 20 g/L, cottonseed meal 10g/lg/L, yeast extract (purchased from Oxiod) 8 g/L, starch 40g/L, glucose 5-10 g/L, KH 2 P0 4 1.5 g/L, MgS0 4 ⁇ 73 ⁇ 40 0.4 g/L, trace element 10 ml/L, initial pH 5.3. Sterilize at 121 ° C for 20 minutes. Glucose was sterilized separately at 115 ° C for 20 minutes.

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Description

一种肽类抗生素的高产菌株及其制备方法和用途 技术领域
本发明涉及抗生素制备领域， 尤其涉及一种肽类抗生素的高产菌株及其 制备方法和用途。 背景技术
在过去的几十年时间里， 严重危害人类健康的真菌感染， 无论是其发生 频率还是感染种类都在不断增加， 特别是在免疫抑制患者中。 而在这段时间 里， 临床上应用比较多的两性霉素和咪唑类以及三唑类抗真菌药物也因其神 经毒性大和耐药性等原因在临床实施上有一定的局限性。 新型抗真菌药 物 棘白霉素类抗生素是 20世纪 70年代发现的一组天然产物， 具有类似 的环状多肽核心和不同的脂肪酸侧链的结构特征， 能竞争性地抑制真菌细胞 壁的 β -D-葡聚糖的合成。 棘白霉素类药物优点为毒性低、 杀菌活性强并且 具有优良的药物动力学性质。
棘白菌素家族包括 WF1 1899A、 棘白菌素类、 西洛芬净、 纽莫康定类、 棘 孢曲菌素类、 以及牡仑康定等成员。 其中 米卡芬净 （Mi cafungi n ) 、 棘白 菌素 （echinocandins ) 及纽莫康定 （ pneumocandin ) 类药物是研究的最为 深入并且已经得到临床应用的抗真菌药物。
米卡芬净 （Mi cafungin ) 是一种水溶性脂肽类棘白菌素类抗真菌药， 由鞘茎点霉 CWe ^^a ^pe r 发酵产物经化学修饰衍生而得， 由藤泽公司 开发作为一种广谱的抗真菌药物。 在日本进行的一项对深部真菌感染 （念珠 菌或曲霉菌） 患者进行的开放性研究中， 本品各剂量组平均治疗约 22 天后 成功率达 92%。 一项在美国进行的单中心研究， 使用本品 50〜150mg与或不 与其他抗真菌药物联用， 针对 14例伴有念珠菌血症的肿瘤患者， 结果 12例 中的 1 1 例 （92 % ) 有效。 没有溶血毒性以及较少的药物相互作用也使得这 类药物比传统的抗真菌药物具有更大的优势。 0{CH2)4CH;
Η,Ν
Na03SO
Mi cafung in结构
Colephoma empe tri产生的一类天然抗真菌药物， 如结构式 I 、 II、 III 保藏号为 FERM BP-2635 的 Colephoma empe tri 生产菌结构 I 的生产能 力很低，产量仅有 700mg/L， 工业化生产的成本非常高。
结构式 I 、 II、 III通式
Figure imgf000003_0001
结构式 I R^OH ， R2=0H
结构式 II R!=0H ， R2=H
结构式 III R!=H ， R2=H
因此， 本领域迫切需要提供一株结构式 I产量较高且更符合工业化生产 的遗传性状稳定、 高产的菌株。 发明内容
本发明旨在提供一种 Colephoma empe tri的诱变菌株。
本发明的另一个目的是提供上述诱变菌株的制备方法。
本发明的再一个目的是提供上述诱变菌株的用途。 在本发明的第一方面， 提供了一种 CW A^a ^pe r 的诱变菌株， 以 CGMCC 4129的保藏编号保藏于中国微生物菌种保藏管理委员会普通微生物中 心。 在本发明的第二方面， 提供了一种如上所述的本发明提供的诱变菌株的 制备方法， 所述的方法包
(a) 将保藏号为 FERM BP-2635的 Colephoma empetri的种子液和亚硝 基胍混合， 得到混合液 a;
(b) 将混合液 a和破壁酶混合， 得到原生质体；
(c) 原生质体再生， 得到单菌落； 和
(d) 培养单菌落， 得到如上所述的诱变菌株。 在本发明的第三方面， 提供了一种如上所述的本发明提供的诱变菌株的 用途， 用于制备如结构式 I所示的化合物；
Figure imgf000004_0001
在本发明的第四方面， 提供了一种制备如式 I所示的化合物的方法， 所 述的方法包括步骤： 15-35°C下， 在发酵培养基培养如上所述的本发明提供 的诱变菌株得到如式 I所示的化合物。
在上述制备方法中， 以发酵培养基总体积计， 所述发酵培养基中含有以 下组分： 玉米浆 5-20g/l， 棉籽粉 5-30g/l， 酵母提取物 6-15g/l， 淀粉 10 一 80g/L， 葡萄糖 5-20 g/L， 无机盐 1.5— 15g/L， 微量元素 10— 50g/L; 所 述无机盐选自下述的一种或其混合： 磷酸盐、 硫酸盐。
在上述制备方法中， 以发酵培养基总体积计， 如上所述的本发明提供的 诱变菌株的接种量为 4一 10v/v %。
在上述制备方法中， 所述发酵培养基的初始 PH5. 5 - 6. 5。 据此， 本发明提供了一株结构式 I产量较高且更符合工业化生产的遗传 性状稳定、 高产的菌株。 具体实施方式
发明人意外地发现， 菌株 Colephoma empe tri FERM BP-2635 经过亚硝 基胍 （NTG ) 的诱变， 再利用溶壁酶得到原生质体后， 通过对再生的原生质 体进行筛选可以得到高产突变株 （保藏编号 CGMCC 4129 ) ， 该突变株经过发 酵可以得到高产量的式 I化合物。 在此基础上， 完成了本发明。
新菌株 本发明提供一种生产式 I 化合物的新菌株， 该菌株分类学上属于 Colephoma empe tri, 已保藏在位于北京的中国微生物菌种保藏管理委员会 普通微生物中心， 保藏号为 CGMCC 4129， 保藏日期为 2010年 8月 31 日。
新菌株的制备方法
本发明提供提供一种保藏号为 CGMCC 4129 的新菌株的制备方法， 所述 的方法如下所示的流程进行：
出发菌株一种子液→NTG 诱变处理→溶壁酶去壁制取原生质体一稀释涂 布平皿一挑取单菌落传种斜面一摇瓶初筛一挑出高产菌株一传种斜面一摇 瓶复筛→挑出高产菌株罐上验证并且进行稳定性试验→保藏菌种
具体地， 本发明提供的方法包括步骤：
( a ) 将保藏号为 FERM BP-2635的 Colephoma empe tri的种子液和亚硝 基胍混合， 得到混合液 a ; (b) 将混合液 a和破壁酶混合， 得到原生质体；
(c) 原生质体再生， 得到单菌落； 和
(d) 培养单菌落， 得到新菌株。
在本发明的一个实施例中，新菌株可以通过以下途径得到：摇瓶培养 1-3 天后的 FERM BP-2635种子液 （细胞干重， DCW 5— 30g/l) 中加入适量 NTG， 继续培养 1-2 天后将种子液离心、 洗涤， 沉淀悬浮后利用溶壁酶（购自广东 省微生物研究所）进行细胞破壁得到原生质体。 稀释后的原生质体涂布于高 渗 PDA (马铃薯葡萄糖琼脂） 平板进行培养得到重组细胞单菌落。 对上述单 菌落进行筛选， 得到诱变新菌株。
进一步地， 本发明还提供了将诱变得到的新菌株发酵得到式 I化合物的 方法。
在本发明的一个实施例中， 诱变得到新菌株及新菌株发酵得到式 I化合 物的方法为：
(1) 出发菌株： Colephoma empetri FERM BP-2635
(2) 出发菌株种子培养
将保藏的 FERM BP-2635 菌株甘油管融化后接种于种子培养基中 （装液 fi 50mL/250mL) ， 25-30°C, 200- 300 rpm摇床振荡培养 3天， 至菌丝干重 达到 5— 30g/L左右。
种子培养基组分为： 蔗糖 10-20 g/L， 酵母提取物 4-10 g/L， 大豆蛋白 胨 10- 20 g/L， KH2P04 1.5-2 g/L， MgS04 · 7H20 0.4-1 g/L， 微量元素 10- 50 g/L， 初始 pH值为 5.3-6.0。 121°C灭菌 20 分钟。
微量元素： FeS04 · 7H20 10-20 g/L, MnS04 · H20 10-20 g/L, ZnS04 · 7H20 2-10g/L, CaCl20.7-2.0 g/L, H具 0.56-2.0 g/L, CuCl2 · 2H200.25-2.0 g/L, (NH4) 6Mo7024 · 7H20 0.19-2.0 g/L， 浓盐酸 500 ml/L。
(3) 单菌落分离
出发菌株种子液先经过 NTG诱变处理， 再经过溶壁酶破壁处理， 所得原 生质体再生获得突变菌株。
(4) 诱变菌株筛选
原生质体涂布于高渗 PDA培养基， 培养 10-12天后的单菌落接种于斜面 培养基进行培养。 8-10天后挖块接种于种子培养基（装液量 25 mL/250 mL) ， 25-30°C、 280 rpm摇床振荡培养 6-10天。 种子培养基接种到发酵培养基（装 液量 25 mL/250 mL) 中， 25- 30°C、 200-300 rpm摇床振荡培养 6- 12天。 培 养结束后用甲醇萃取发酵液， 高效液相色谱测定发酵液中式 I化合物含量。
其中所涉及各培养基组成可参见文献 Improvement of FR901379 production by mutant selection and medium optimization , Journal of Bioscience and Bioengineering VOL 107 No.5， 530 - 534， 2009， Journal of antibiotics, Vol 45， No.12, Dec 1992, 1867-1874.
高渗 PDA平板培养基： 马铃薯 300 g/L， 葡萄糖 20 g/L， 琼脂 15 g/L， 蔗糖 273.6 g/L， 121°C灭菌 20 分钟。
(5) 诱变菌株发酵
相关技术方案在文献中有报道，详见
Improvement of FR901379 production by mutant selection and medium optimization， Journal of Bioscience and Bioengineering VOL 107 No.5， 530 - 534 ， 2009 , Journal of antibiotics, Vol 45， No.12, Dec 1992, 1867-1874. 本发明提到的上述特征， 或实施例提到的特征可以任意组合。 本案说明书 所揭示的所有特征可与任何组合物形式并用， 说明书中所揭示的各个特征， 可 以任何可提供相同、 均等或相似目的的替代性特征取代。 因此除有特别说明， 所揭示的特征仅为均等或相似特征的一般性例子。 本发明的主要优点在于：
1、 获得了一株高产且遗传性状稳定的诱变新菌株。
2、 由于新菌株遗传稳定性好， 且产生的杂质较少， 有利于式 I 化合物 生产中产物分离纯化， 和工艺放大， 因此适合工业化生产。
3、 在优化条件下发酵生产式 I化合物， 产量可达到 1.5g/L。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于 说明本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实 验方法， 通常按照常规条件或按照制造厂商所建议的条件。 除非另外说明， 否则所有的百分数、 比率、 比例、 或份数按重量计。
本发明中的重量体积百分比中的单位是本领域技术人员所熟知的， 例如 是指在 100毫升的溶液中溶质的重量。
除非另行定义， 文中所使用的所有专业与科学用语与本领域熟练人员所 熟悉的意义相同。 此外， 任何与所记载内容相似或均等的方法及材料皆可应 用于本发明方法中。 文中所述的较佳实施方法与材料仅作示范之用。 本发明实施例中发酵液中式 I 化合物的含量的高效液相色谱法测定方 法：
采用高效液相色谱法测定发酵液中式 I化合物的含量：
色谱柱： Calesil DOS- 100 (4.6mmX250 mm, 5 μ m)，
流动相： 乙腈： 水 = 50: 50， 含 0.05N磷酸二氢铵
柱温 35°C
等梯度洗脱， 流速为 1.0 mL/分钟，
进样量 5 L， 检测波长 210nm。 实施例 1
诱变得到新菌株 CGMCC 4129
1. 诱变
将保藏的 FERM BP-2635菌株甘油管融化后以 4%的接种量接种于种子培 养基中 （装液量 50mL/250mL) ， 25。C、 280 rpm摇床振荡培养 2天， 至菌丝 干重达到 5-30 g/L左右。 在种子液中以 10 μ g/mL的浓度加入诱变剂 NTG, 继续培养 1天。 培养 1天后， 取含 NTG的种子液 10mL，5000 rpm离心 10 分 钟，所得沉淀用两倍体积的 0.6M的 NaCl洗涤两次以除去培养基和 NTG， 此诱 变过程致死率为 85-90%。
种子培养基：蔗糖 10 g/L，酵母提取物 5 g/L,大豆蛋白胨 10 g/L， KH2P04 1.5 g/L, MgS04 · 7¾0 0.4 g/L， 微量元素 10 g/L， 初始 pH值为 5.3。 121°C 灭菌 20 分钟。
微量元素： FeS04 · 7¾0 10 g/L, MnS04 · H20 10 g/L, ZnS04 · 7H20 2 g/L, CaCl2 0.7 g/L, H3BO30.56 g/L, CuCl2 · 2H200.25 g/L, (NH4) 6Mo7024 · 7H20 0.19 g/L, 浓盐酸 500 ml/Lo
2.原生质体制备及单菌落分离
洗后菌丝加入含有 20mg/mL 的溶壁酶 （2000单位 /mg) ， 10mg/ml 蜗牛 酶 （5单位 /mg ) 和 10mg/ml纤维素酶 （15单位 /mg ) 的酶混合液 （0. 5MNaCl 磷酸氢二钠 -柠檬酸缓冲液溶解（PH6. 0 ) ) 10mL， 30°C下 80rpm震荡酶解 5h。 用棉花过滤酶解后的反应液以除去菌丝得到只含有原生质体的单细胞悬浮 液。 取 1 mL此溶液 14000 rpm离心 10 分钟，沉淀用 1 mL的含 0. 5MNaCl磷 酸氢二钠-柠檬酸缓冲液 （pH6. 0 ) 溶解。 将此液稀释成不同倍数均匀涂布于 含 0. 8M蔗糖的高渗 PDA培养基 25°C培养 6-8天后获得约 6000个单菌落。
3.高产菌株 CGMCC 4129的筛选过程
挑选培养 8 天后的单菌落接种于斜面培养基进行培养。 8 天后挑取 0. 5-1. Ocm2面积的菌落接种于种子培养基（装液量 25 mL/250 mL )，共计 4000 个单菌落， 25°C、 280 rpm摇床振荡培养 5天。 种子培养基按 4 %接种量接种 到发酵培养基 （装液量 25 mL/250 mL ) 中， 25°C、 280 rpm摇床振荡培养 10 天 （培养至 6天时补加 5%淀粉） 。
培养结束后用 50ml 甲醇萃取发酵液， 高效液相色谱测定发酵液中式 I 化合物含量，其中共有 5株高产菌株。其中一株所得到的高产菌株 CGMCC 4129 经过复筛， 式 I化合物的产量达到 1. 4g/L。 实施例 2
新菌株 CGMCC 4129生产式 I化合物
将种子培养基按 4 %接种量将实施例 1得到的新菌株 CGMCC 4129接种到 发酵培养基中， 在 50升发酵罐中进行培养， 培养温度 25°C， 发酵液 pH维持 在 6. 5。 培养 10天后， 式 I化合物的产量达到 1. 5g/L (培养至 6天时补加 5%淀粉） 。
发酵培养基玉米浆 20 g/L， 棉籽粉 10g/l g/L， 酵母提取物 （购自 Ox i od 公司） 8 g/L， 淀粉 40g/L， 葡萄糖 5- 10 g/L， KH2P04 1. 5 g/L， MgS04 · 7¾0 0. 4 g/L， 微量元素 10ml/L， 初始 pH值为 5. 3。 121 °C灭菌 20分钟。 葡萄糖 单独灭菌 1 15°C， 20分钟。
微量元素： FeS04 · 7¾0 10 g/L , MnS04 · H20 10 g/L , ZnS04 · 7H20 2 g/L , CaCl2 0. 7 g/L， H3BO3 0. 56 g/L， CuCl2 · 2H20 0. 25 g/L， ( NH4 ) 6Mo7024 · 7H20 0. 19 g/L， 浓盐酸 500 ml/L o 对比例
用以下方法比较出发菌株 FERM BP-2635与突变株 CGMCC 4129产式 I化 合物的能力：
用实施例 2的培养方法分别培养出发菌株和突变株， 培养结束后用两倍 体积的甲醇萃取发酵液， 高效液相色谱测定发酵液中式 I化合物含量。 结果 表 1
Figure imgf000010_0001
其中所涉及各培养基组成如下:
筛选培养基： 马铃薯 300 g/L， 葡萄糖 20g/L， 琼脂 15 g/L， 蔗糖 273.6 g/L， 121°C灭菌 20 分钟。
斜面培养基： 马铃薯 300 g/L， 葡萄糖 20 g/L， 琼脂 15 g/L， 121°C 灭菌 20 分钟。
种子培养基：蔗糖 10 g/L，酵母提取物 5 g/L，大豆蛋白胨 10 g/L， KH2P04 1.5 g/L， MgS04 · 7¾0 0.4 g/L， 微量元素 10 g/L， 初始 pH值为 5.3。 121°C 灭菌 20 分钟。
发酵培养基：玉米浆 20 g/L， 棉籽粉 10g/l g/L，酵母提取物（购自 Oxiod 公司） 8 g/L， 淀粉 40g/L， 葡萄糖 5- 10 g/L， KH2P04 1.5 g/L， MgS04 · 7¾0 0.4 g/L， 微量元素 10ml/L， 初始 pH值为 5.3。 121°C灭菌 20分钟。 葡萄糖 单独灭菌 115°C， 20分钟。
微量元素： FeS04 · 7¾0 10 g/L, MnS04 · H20 10 g/L, ZnS04 · 7H202 g/L,
CaCl20.7 g/L， H3BO30.56 g/L， CuCl2 · 2H200.25 g/L， (NH4) 6Mo7024 · 7H20 0.19 g/L， 浓盐酸 500 ml/Lo 实施例 3
新菌株 CGMCC 4129稳定性
采用如实施例 2的培养方式及培养基组成， 进行传代培养。 结果见表 2。 表 2 新菌种传代稳定性 传代次数 F1 F2 F6 式 I化合物产量 （g/L ) 1. 5 1. 3 1. 6 结果表明， 新菌株稳定性好。 以上所述仅为本发明的较佳实施例而已， 并非用以限定本发明的实质技 术内容范围， 本发明的实质技术内容是广义地定义于申请的权利要求范围 中， 任何他人完成的技术实体或方法， 若是与申请的权利要求范围所定义的 完全相同， 也或是一种等效的变更， 均将被视为涵盖于该权利要求范围之中。

Claims

权 利 要 求
1.一种 Colephoma empetri的诱变菌株， 以 CGMCC 4129 的保藏编号保 藏于中国微生物菌种保藏管理委员会普通微生物中心。
2.—种如权利要求 1所述的诱变菌株的制备方法， 其特征在于， 所述的 方法包括步骤：
(a) 将保藏号为 FERM BP-2635的 Colephoma empetri的种子液和亚硝 基胍混合， 得到混合液 a;
(b) 将混合液 a和破壁酶混合， 得到原生质体；
(c) 原生质体再生， 得到单菌落； 和
(d) 培养单菌落， 得到如权利要求 1所述的诱变菌株。
3.—种如权利要求 1所述的诱变菌株的用途， 其特征在于， 用于制备如 结构式 I所示的化合物；
Figure imgf000012_0001
4.一种制备如式 I所示的化合物的方法， 其特征在于， 所述的方法包括 步骤： 15-35°C下， 在发酵培养基培养如权利要求 1 所述的诱变菌株得到如 式 I所示的化合物。
5.如权利要求 4所述的制备方法，其特征在于， 以发酵培养基总体积计， 所述发酵培养基中含有以下组分： 玉米浆 5-20g/l， 棉籽粉 5-30g/l， 酵母 提取物 6-15g/l， 淀粉 10— 80g/L， 葡萄糖 5-20 g/L， 无机盐 1. 5— 15g/L， 微量元素 10— 50g/L。
6.如权利要求 4所述的制备方法， 其特征在于， 所述无机盐选自下述的 一种或其混合： 磷酸盐、 硫酸盐。
7.如权利要求 4所述的制备方法，其特征在于， 以发酵培养基总体积计， 如权利要求 1所述的诱变菌株的接种量为 4一 10v/v %。
8.如权利要求 4所述的制备方法， 其特征在于， 所述发酵培养基的初始 pH5. 5— 6. 5。
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CN102533551A (zh) 2012-07-04
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