CN110305926B - 一种基于两性霉素b代谢途径的发酵方法 - Google Patents
一种基于两性霉素b代谢途径的发酵方法 Download PDFInfo
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- 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
- C12N1/20—Bacteria; Culture media therefor
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- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种基于两性霉素B代谢途径的发酵方法,所述方法包括:将结节链霉菌接种至发酵培养基,25~30℃、150~250r/min条件下培养96~168h,在发酵培养第12~36h内向发酵液中添加关键差异代谢物质,发酵结束后,发酵产物经分离纯化获得所述的两性霉素B。通过在AMB发酵培养至12~36h添加各种关键差异代谢物,来提高AMB的发酵水平。从本发明实施例中可以看出,发酵培养24h添加丙酮酸可以使AMB的产量由5360mg/L提高至6645mg/L;在培养基中添加D‑泛酸钙或烟酰胺可使AMB产量分别提高18.6%和12.5%。
Description
(一)技术领域
本发明涉及一种基于两性霉素B代谢途径的发酵方法,属于微生物发酵工程技术领域。
(二)背景技术
两性霉素B(Amphotericin B,AMB)是结节链霉菌产生的一种次级代谢产物,是一种七烯大环内脂类抗生素。其抑菌机理主要是其可与敏感真菌胞浆膜中的固醇结合,在脂质双分子层中形成的亲水孔道,从而改变胞浆膜的通透性,致使细胞内物质如电解质离子、核苷酸、氨基酸和蛋白质分子渗漏,破坏细胞的正常代谢,杀灭真菌。多烯大环内酯类抗生素的这一作用取决于固醇的结构、含量及其与脂质分子的排列关系。哺乳动物细胞的胞浆膜中含有固醇,所以多烯大环内酯类抗生素对动物和人都有毒性。由于真菌膜中主要含麦角固醇,动物膜中主要含胆固醇,两性霉素B对麦角固醇的亲和力大于对胆固醇的亲和力,因而对人的毒性亦较小。
两性霉素B生物合成的调控机制并不清晰,综合文献可知,两性霉素B的内酯环是由16个乙酸单元、3个丙酸单元和一个海藻糖胺基团缩合形成,而乙酸单元和丙酸单元分别以丙二酸单酰辅酶A和甲基丙二酸单酰辅酶A为前体的。这些前体涉及多种主要代谢途径,如糖酵解(EMP),戊糖磷酸途径,三羧酸循环(TCA),脂肪酸,莽草酸和氨基酸代谢。因此,与上述途径相关的细胞内代谢物的变化曲线对于揭示更多的两性霉素B生物合成的潜在关键因素是至关重要的。目前两性霉素B的产量还较低,且不稳定,受培养条件和环境等因素的影响很大。
代谢组学专注于生物系统中的综合代谢物分析,可以为细胞代谢提供独特的见解。许多研究人员利用它来识别代谢物在发酵过程中枯竭或积累,从而制定优化流程和策略以增加产品滴度。目前还没有基于代谢组学和代谢途径分析方法在结节链霉菌中的应用。
(三)发明内容
本发明目的是一种基于两性霉素B代谢途径的发酵方法,有效提升AmB的产量。
本发明采用的技术方案是:
一种基于两性霉素B代谢途径的发酵方法,所述方法包括:将结节链霉菌接种至发酵培养基,25~30℃、150~250r/min条件下培养96~168h,在发酵培养第12~36h内向发酵液中添加关键差异代谢物质,发酵结束后,发酵产物经分离纯化获得所述的两性霉素B;所述的关键差异代谢物为下列之一或其中两种以上的混合物:D-泛酸钙、丙酮酸、甘氨酸、海藻糖、烟酰胺、甘油磷酸钙、肌苷、卵磷脂、S-腺苷蛋氨酸(SAM)。
所述D-泛酸钙、海藻糖、烟酰胺、甘油磷酸钙、肌苷的添加浓度范围各自独立为0.001~1g/L,丙酮酸的添加浓度为0.01~10mL/L,卵磷脂的添加浓度为0.01~20g/L,甘氨酸的添加浓度为0.1~10mmol/L,S-腺苷蛋氨酸的添加浓度为0.001~5mmol/L。
具体的,所述发酵培养基组成如下:葡萄糖60~80g/L,棉籽粉10~30g/L,碳酸钙5~15g/L,磷酸二氢钾0.05~5g/L,溶剂为水,pH为7.0~7.2。
通常,所述结节链霉菌在发酵培养前先进行种子培养,再将种子液以体积浓度2~10%的接种量接种至发酵培养基,所述种子培养基组成如下:蛋白胨10~30g/L,酵母提取物5~20g/L,氯化钠1~10g/L,葡萄糖5~20g/L,碳酸钙0.5~2g/L,溶剂为水,pH为7.0~7.2。将结节链霉菌接种于种子培养基中,在25~30℃、150~250r/min条件下培养40~72h,得到种子液。
优选的,所述关键差异代谢物为丙酮酸(添加量优选为0.1%,v/v)、D-泛酸钙(添加量优选为0.0025%,w/v,即0.025g/L)或烟酰胺(添加量优选为0.0025%,w/v,即0.025g/L)。
发酵液中两性霉素B含量检测采用HLPC法。样品预处理:取150μL发酵液置于2mL离心管中,将发酵液与二甲基亚砜按体积比1:9混合,12000rpm离心5min,收集上清液采用0.45μm微滤膜过滤,滤液采用伊利特HPLC分析。HPLC流动相配制:准确称取1.1g乙二胺四乙酸二钠和4.1g无水乙酸钠,用超纯水溶解并定容至1000mL,0.45μm微滤膜过滤;滤液与色谱纯乙腈和甲醇按体积比9:7:4混合,并采用超声波脱除气体。HPLC分析条件:伊利特HPLC泵,UV3100紫外-可见光检测器,色谱柱为Unitary C18(4.6×250mm),流动相比例采用盐溶液:乙腈:甲醇=9:7:4,流速为1.0mL/min,紫外检测方法为双波长检测,紫外检测波长分别为304nm和405nm,其中304nm处为AMA,405nm处为AMB,进样量为20μL,柱温25℃。
本发明通过分析代谢途径中的各种关键差异代谢物以及其介导的代谢调节机制,并以这些关键差异代谢物作为外源添加物质加入到发酵培养基中,提高了两性霉素B的产量。
与现有技术相比,本发明的有益效果主要体现在:通过在AMB发酵培养至12~36h添加各种关键差异代谢物,来提高AMB的发酵水平。从本发明实施例中可以看出,发酵培养24h添加丙酮酸可以使AMB的产量由5360mg/L提高至6645mg/L;在培养基中添加D-泛酸钙或烟酰胺可使AMB产量分别提高18.6%和12.5%。
(四)附图说明
图1为D-泛酸钙的不同添加浓度对AMB产量的影响;
图2为丙酮酸的不同添加浓度对AMB产量的影响;
图3为甘氨酸的不同添加浓度对AMB产量的影响;
图4为海藻糖的不同添加浓度对AMB产量的影响;
图5为烟酰胺的不同添加浓度对AMB产量的影响;
图6为甘油磷酸钙的不同添加浓度对AMB产量的影响;
图7为肌苷的不同添加浓度对AMB产量的影响;
图8为卵磷脂的不同添加浓度对AMB产量的影响;
图9为SAM的不同添加浓度对AMB产量的影响;
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
对比例:
本发明实施例中所用的菌种为结节链霉菌Streptomyces nodosus ZJB2016050(CCTCC No:M 2017426),来自中国典型培养物保藏中心。
两性霉素B的发酵生产方法,包括以下步骤:
(1)Streptomyces nodosus ZJB2016050种子培养:
从GYM斜面培养基上取一环带灰色孢子菌落接种于一瓶含有50mL种子培养基的250mL三角瓶中,培养温度为25℃,摇床转速200r/min,培养2天,获得种子液;所述种子培养基组成如下:蛋白胨15g,酵母提取物10g,氯化钠5g,葡萄糖10g,碳酸钙1g,蒸馏水补足至1L,以NaOH调节pH值为7.0,115℃灭菌30min;
(2)Streptomyces nodosus ZJB2016050发酵培养:
将2mL种子液接种到50mL发酵培养基中,发酵容器为250mL三角瓶,在25℃、200r/min条件下摇床培养5天;所述发酵培养基组成如下:葡萄糖70g,棉籽粉25g,碳酸钙9g,磷酸二氢钾0.1g,蒸馏水补足至1L,pH为7.0,115℃灭菌30min。
在发酵培养第5天时结束发酵,取样检测AMB含量,用高效液相色谱测得AMB含量为5360mg/L。
实施例1
基于两性霉素B代谢途径的发酵生产方法,包括以下步骤:
(1)Streptomyces nodosus ZJB2016050种子培养:
从GYM斜面培养基上取一环带灰色孢子菌落接种于一瓶含有50mL种子培养基的250mL三角瓶中,培养温度为25℃,摇床转速200r/min,培养2天,获得种子液;所述种子培养基如对比例所述。
(2)Streptomyces nodosus ZJB2016050发酵培养:
将2mL种子液接种到50mL发酵培养基中,发酵容器为250mL三角瓶,并在发酵24h时,在发酵液中按照0.001%~0.02%(w/v)的量添加D-泛酸钙,在25℃、200r/min条件下摇床培养5天;所述发酵培养基如对比例所述。
在发酵培养第5天时结束发酵,取样检测AMB含量。本发明考察了D-泛酸钙不同添加浓度对AMB产量的影响,用高效液相色谱测得AMB含量为6356mg/L,结果如图1所示,结果表明在添加浓度为0.0025%时,添加效果最好,两性霉素B的产量与对照组(5360mg/L)相比,提高了18.6%。
实施例2:
基于两性霉素B代谢途径的发酵生产方法,包括以下步骤:
(1)Streptomyces nodosus ZJB2016050种子培养:
从GYM斜面培养基上取一环带灰色孢子菌落接种于一瓶含有50mL种子培养基的250mL三角瓶中,培养温度为25℃,摇床转速200r/min,培养2天,获得种子液;所述种子培养基如对比例所述。
(2)Streptomyces nodosus ZJB2016050发酵培养:
将2mL种子液接种到50mL发酵培养基中,发酵容器为250mL三角瓶,并在发酵24h时,在发酵液中按照0.01%~0.1%(v/v)的量添加丙酮酸,在25℃、200r/min条件下摇床培养5天;所述发酵培养基如对比例所述。
在发酵培养第5天时结束发酵,取样检测AMB含量。由于丙酮酸是中心糖代谢的重要代谢物,本发明还考察了丙酮酸不同添加浓度对AMB产量的影响,用高效液相色谱检测发酵液中AMB含量,结果如图2所示,结果表明在添加浓度为0.1%时,添加效果最好,两性霉素B的产量为6645mg/L,与对照组(5360mg/L)相比,提高了24%。
实施例3:
基于两性霉素B代谢途径的发酵生产方法,包括以下步骤:
(1)Streptomyces nodosus ZJB2016050种子培养:
从GYM斜面培养基上取一环带灰色孢子菌落接种于一瓶含有50mL种子培养基的250mL三角瓶中,培养温度为25℃,摇床转速200r/min,培养2天,获得种子液;所述种子培养基如对比例所述。
(2)Streptomyces nodosus ZJB2016050发酵培养:
将2mL种子液接种到50mL发酵培养基中,发酵容器为250mL三角瓶,并在发酵24h时,在发酵液中按照0.25-4mmol/L的终浓度添加甘氨酸,在25℃、200r/min条件下摇床培养5天;所述发酵培养基如对比例所述。
在发酵培养第5天时结束发酵,取样检测AMB含量。由于甘氨酸属于叶酸生物合成途径的重要中间代谢物,本发明还考察了甘氨酸不同添加终浓度对AMB产量的影响,用高效液相色谱检测发酵液中AMB含量,结果如图3所示,结果表明在添加浓度为0.5mmol/L时,添加效果最好,两性霉素B的产量为5986mg/L,与对照组(5360mg/L)相比,提高了10%。
实施例4:
基于两性霉素B代谢途径的发酵生产方法,包括以下步骤:
(1)Streptomyces nodosus ZJB2016050种子培养:
从GYM斜面培养基上取一环带灰色孢子菌落接种于一瓶含有50mL种子培养基的250mL三角瓶中,培养温度为25℃,摇床转速200r/min,培养2天,获得种子液;所述种子培养基如对比例所述。
(2)Streptomyces nodosus ZJB2016050发酵培养:
将2mL种子液接种到50mL发酵培养基中,发酵容器为250mL三角瓶,并在发酵24h时,在发酵液中按照0.001%-0.02%(w/v)的量添加海藻糖,在25℃、200r/min条件下摇床培养5天;所述发酵培养基如对比例所述。
在发酵培养第5天时结束发酵,取样检测AMB含量。本发明还考察了海藻糖不同添加浓度对AMB产量的影响,用高效液相色谱检测发酵液中AMB含量,结果如图4所示,结果表明在添加浓度为0.005%时,添加效果同对照组(5360mg/L)相近,两性霉素B的产量为5346mg/L。
实施例5:
基于两性霉素B代谢途径的发酵生产方法,包括以下步骤:
(1)Streptomyces nodosus ZJB2016050种子培养:
从GYM斜面培养基上取一环带灰色孢子菌落接种于一瓶含有50mL种子培养基的250mL三角瓶中,培养温度为25℃,摇床转速200r/min,培养2天,获得种子液;所述种子培养基如对比例所述。
(2)Streptomyces nodosus ZJB2016050发酵培养:
将2mL种子液接种到50mL发酵培养基中,发酵容器为250mL三角瓶,并在发酵24h时,在发酵液中按照0.001%-0.02%(w/v)的量添加烟酰胺,在25℃、200r/min条件下摇床培养5天;所述发酵培养基如对比例所述。
在发酵培养第5天时结束发酵,取样检测AMB含量。由于烟酰胺是结节链霉菌生产AMB过程中的重要中间代谢物,本发明还考察了烟酰胺不同添加浓度对AMB产量的影响,用高效液相色谱检测发酵液中AMB含量,结果如图5所示,结果表明在添加浓度为0.0025%时,添加效果最好,两性霉素B的产量为6030mg/L,对照组(5360mg/L)相比,提高了12.5%。
实施例6:
基于两性霉素B代谢途径的发酵生产方法,包括以下步骤:
(1)Streptomyces nodosus ZJB2016050种子培养:
从GYM斜面培养基上取一环带灰色孢子菌落接种于一瓶含有50mL种子培养基的250mL三角瓶中,培养温度为25℃,摇床转速200r/min,培养2天,获得种子液;所述种子培养基如对比例所述。
(2)Streptomyces nodosus ZJB2016050发酵培养:
将2mL种子液接种到50mL发酵培养基中,发酵容器为250mL三角瓶,并在发酵24h时,在发酵液中按照0.001%-0.02%(w/v)的量添加甘油磷酸钙,在25℃、200r/min条件下摇床培养5天;所述发酵培养基如对比例所述。
在发酵培养第5天时结束发酵,取样检测AMB含量。由于甘油磷酸是甘油脂代谢的重要中间代谢物,采用相应的甘油磷酸钙盐作为外源添加物质,本发明还考察了甘油磷酸钙不同添加浓度对AMB产量的影响,用高效液相色谱检测发酵液中AMB含量,结果如图6所示,结果表明添加效果同对照组(5360mg/L)相近,两性霉素B的产量为5462mg/L。
实施例7:
基于两性霉素B代谢途径的发酵生产方法,包括以下步骤:
(1)Streptomyces nodosus ZJB2016050种子培养:
从GYM斜面培养基上取一环带灰色孢子菌落接种于一瓶含有50mL种子培养基的250mL三角瓶中,培养温度为25℃,摇床转速200r/min,培养2天,获得种子液;所述种子培养基如对比例所述。
(2)Streptomyces nodosus ZJB2016050发酵培养:
将2mL种子液接种到50mL发酵培养基中,发酵容器为250mL三角瓶,并在发酵24h时,在发酵液中按照0.001%-0.02%(w/v)的量添加肌苷,在25℃、200r/min条件下摇床培养5天;所述发酵培养基如对比例所述。
在发酵培养第5天时结束发酵,取样检测AMB含量。由于肌苷是结节链霉菌生产AMB过程中的重要代谢物,本发明还考察了肌苷的不同添加浓度对AMB产量的影响,用高效液相色谱检测发酵液中AMB含量,结果如图7所示,结果表明在添加浓度为0.0025%时,添加效果最好,两性霉素B的产量为5569mg/L,对照组(5360mg/L)相比,提高了4.3%。
实施例8:
基于两性霉素B代谢途径的发酵生产方法,包括以下步骤:
(1)Streptomyces nodosus ZJB2016050种子培养:
从GYM斜面培养基上取一环带灰色孢子菌落接种于一瓶含有50mL种子培养基的250mL三角瓶中,培养温度为25℃,摇床转速200r/min,培养2天,获得种子液;所述种子培养基如对比例所述。
(2)Streptomyces nodosus ZJB2016050发酵培养:
将2mL种子液接种到50mL发酵培养基中,发酵容器为250mL三角瓶,并在发酵24h时,在发酵液中按照0.01%-1%(w/v)的量添加卵磷脂,在25℃、200r/min条件下摇床培养5天;所述发酵培养基如对比例所述。
在发酵培养第5天时结束发酵,取样检测AMB含量。由于卵磷脂是甘油磷脂代谢过程的重要中间代谢物,本发明还考察了卵磷脂的不同添加浓度对AMB产量的影响,用高效液相色谱检测发酵液中AMB含量,结果如图8所示,结果显示两性霉素B的产量与对照组(5360mg/L)相比,产量没有提升,有所下降。
实施例9:
基于两性霉素B代谢途径的发酵生产方法,包括以下步骤:
(1)Streptomyces nodosus ZJB2016050种子培养:
从GYM斜面培养基上取一环带灰色孢子菌落接种于一瓶含有50mL种子培养基的250mL三角瓶中,培养温度为25℃,摇床转速200r/min,培养2天,获得种子液;所述种子培养基如对比例所述。
(2)Streptomyces nodosus ZJB2016050发酵培养:
将2mL种子液接种到50mL发酵培养基中,发酵容器为250mL三角瓶,并在发酵24h时,在发酵液中按照0.001%-0.02%(v/v)的量添加SAM,在25℃、200r/min条件下摇床培养5天;所述发酵培养基如对比例所述。
在发酵培养第5天时结束发酵,取样检测AMB含量。作为蛋白质,核酸的甲基供体酸和多糖,SAM不仅参与其中转移硫基和核糖基,也有调节作用细胞内代谢物参与原发性和继发性代谢。同时由于其生理活性和生物学功能,微生物中SAM水平的微小变化细胞对细胞生长和代谢产物合成具有显着影响。因此本发明还考察了SAM的不同添加浓度对AMB产量的影响,用高效液相色谱检测发酵液中AMB含量,结果如图9所示,结果表明在添加浓度为0.01mmol/L时,添加效果最好,两性霉素B的产量为5641mg/L,对照组(5012mg/L)相比,提高了12.5%。
Claims (3)
1.一种基于两性霉素B代谢途径的发酵方法,所述方法包括:将结节链霉菌接种至发酵培养基,25~30℃、150~250r/min条件下培养96~168h,在发酵培养第12~36h内向发酵液中添加关键差异代谢物质,发酵结束后,发酵产物经分离纯化获得所述的两性霉素B;所述的关键差异代谢物为丙酮酸、D-泛酸钙或烟酰胺;所述丙酮酸添加量为1mL/L;所述D-泛酸钙或烟酰胺添加量为0.025g/L。
2.如权利要求1所述的方法,其特征在于所述发酵培养基组成如下:葡萄糖60~80g/L,棉籽粉10~30g/L,碳酸钙5~15g/L,磷酸二氢钾0.05~5g/L,溶剂为水,pH为7.0~7.2。
3.如权利要求2所述的方法,其特征在于所述结节链霉菌在发酵培养前先进行种子培养,再将种子液以体积浓度2~10%的接种量接种至发酵培养基,所述种子培养基组成如下:蛋白胨10~30g/L,酵母提取物5~20g/L,氯化钠1~10g/L,葡萄糖5~20g/L,碳酸钙0.5~2g/L,溶剂为水,pH为7.0~7.2。
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