CN107893048B - 一种产两性霉素b的重组结节链霉菌及其应用 - Google Patents
一种产两性霉素b的重组结节链霉菌及其应用 Download PDFInfo
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Abstract
本发明公开了一种产两性霉素B的重组结节链霉菌及其应用，所述重组结节链霉菌是将SEQ ID NO.1所示卡那霉素抗性基因序列导入结节链霉菌(Streptomyces nodosus)ZJB2016050获得的；通过导入卡那霉素抗性基因，使原本不具抗性的生产菌株带有卡那霉素抗性，提高种子培养基的菌种纯度和质量，实验室种子培养未出现染菌现象。通过表达功能基因透明颤菌血红蛋白(vhb)，提高AmB产量约15％；通过表达功能基因S‑腺苷甲硫氨酸合成酶基因(metk)，提高AmB产量约40％。
Description
(一)技术领域
本发明涉及一种两性霉素B的生产方法，特别涉及一种高产两性霉素B的重组结节链霉菌及其应用。
(二)背景技术
两性霉素B(Amphotericin B，AmB)是一种多烯类广谱抗真菌抗生素，由结节链霉菌(Streptomyces nodosus)产生，其菌株于1955年从委内瑞拉奥里诺科河三角洲的土样中收集分离获得，并发现其具有抗真菌活性。1959年，在结节链霉菌发酵液中提取获得AmB。1966年开始上市，是第一个用于深部真菌感染的药物，已使用近半个世纪，目前仍然是临床上不可替代的抗真菌药物。AmB属于多稀大环内酯类抗生素，具有广谱性的对真菌抗性，特别是对于具有生命威胁性的全身性真菌感染例如白色念珠菌，曲霉属真菌等，同时还具有有效的抗病毒、寄生虫药性，例如阮病毒、利什曼原虫等。AmB多用于免疫系统损伤或免疫力较差患者，如器官移植接受者，HIV患者，以及使用抑制免疫力药物的肿瘤患者等。
AmB分子式C47H73NO17，结构式如图1，熔点大于170℃，旋光度+420，在405、382、362、345、283、273、263、225mm紫外波长下具有吸收峰。
AmB为黄色或橙黄色粉末，无味，有引湿性，在日光下易破坏失效。在二甲基亚砜中溶解，在二甲基甲酰胺中微溶，在甲醇中极微溶解，在水，无水乙醇，三氯甲烷或乙醚中基本不溶，(pH 6-7)均少于1mg/L。
AmB的抗真菌作用机制为：AmB能与真菌细胞膜上的麦角固醇结合，在膜上形成微孔，使膜的通透性发生改变，最终导致重要的细胞内容物K离子、核苷酸、氨基酸等无节制的流失而造成菌体死亡。然而AmB也会在较小程度上与哺乳动物细胞膜上的胆固醇相互作用，造成一定的副作用，特别是肾毒性。虽然其具有一定的副作用，但AmB仍然是目前治疗人类深部全身性真菌感染的最重要一种抗生素。人们加强了对AmB新给药形式的研究，例如在20世纪90年代出现的Abelcet、Ambisome脂质体药物；Amphocil胶状悬浮液药物等AmB脂质体、乳剂、纳米粒子等新形式药物，为了降低毒性，提高药物到达作用区域的效率。
现有的产AmB的菌株多为野生型筛选高产菌株，均无抗生素抗性，因此在工业发酵过程中会出现杂菌污染现象。卡那霉素(Kanamycin)是一种由卡那链霉菌(Streptomycekanamyceticus)产生的氨基糖苷类抗生素，其具有抗菌谱广、杀菌作用强、价格优廉等特点。其主要作为一种蛋白质生物合成抑制剂，通过与30S核糖体结合从而致使mRNA密码翻译出现差错，进而致使细胞死亡。通过表达氨基糖苷磷酸转移酶，即卡那霉素抗性基因，可使菌种带有卡那霉素抗性。将卡那霉素抗性基因(kan)导入结节链霉菌(Streptomycesnodosus)，并在发酵生产过程中加入抗生素卡那霉素，能够降低染杂菌的概率，提升上罐发酵成功率，尤其在工业生产当中，能够降低上罐发酵的成本。
部分功能基因对次级代谢产物表达具有正向作用。metk为S-腺苷甲硫氨酸(SAM)合成酶编码基因。SAM合成酶在细菌、真菌、放线菌中均有发现，细胞内的SAM对于大多数链霉菌中分化与抗生素生产等具有重要作用。SAM可作为次级代谢产物的甲基供体，是嘧啶、腺苷、以及多种蛋白、小分子物质甲基化的来源，还可作为抗生素生产基因的转录激活因子。vhb透明颤菌血红蛋白，能够促进微生物细胞胞内氧的传输，提高氧的利用率，在有氧发酵过程中具有积极作用。
(三)发明内容
本发明目的是提供一种带有抗生素抗性(卡那霉素)的产两性霉素B(AmB)结节链霉菌及其构建方法和应用，通过在结节链霉菌中表达外源基因即卡那霉素抗性基因，使结节链霉菌带有卡那霉素抗性，抵抗外界细菌污染，降低发酵不稳定现象，进而提高生产效率和产量；通过过表达透明颤菌血红蛋白(vhb)基因及S-腺苷甲硫氨酸合成酶基因(metk)，进一步提升结节链霉菌合成AmB的能力，有效提升AmB的产量。
本发明采用的技术方案是：
本发明提供一种产两性霉素B的重组结节链霉菌，所述重组结节链霉菌是将SEQID NO.1所示卡那霉素抗性基因序列(氨基酸序列为SEQ ID NO.2所示)导入结节链霉菌(Streptomyces nodosus)ZJB2016050获得的；所述结节链霉菌(Streptomyces nodosus)ZJB2016050保藏于中国典型培养物保藏中心(CCTCC)，保藏日期2017年7月17号，保藏编号CCTCC NO：M 2017426，保藏地址为中国武汉，武汉大学，邮编430072。
进一步，所述重组结节链霉菌还包含SEQ ID NO.7或SEQ ID NO.8所示外源基因，即将外源基因导入高产AmB的重组结节链霉菌。
进一步，所述重组结节链霉菌是将SEQ ID NO.1、SEQ ID NO.7(透明颤菌血红蛋白基因)所示基因序列导入结节链霉菌(Streptomyces nodosus)ZJB2016050获得的。
进一步，所述重组结节链霉菌是将SEQ ID NO.1、SEQ ID NO.8(S-腺苷甲硫氨酸合成酶基因)所示基因序列导入结节链霉菌(Streptomyces nodosus)ZJB2016050获得的。
本发明还提供一种所述产AmB的重组结节链霉菌在提高AmB产量中的应用。
进一步，所述应用是将产AmB的重组结节链霉菌接种至发酵培养基，25-30℃(优选28℃)，220rpm发酵培养，获得含AmB的发酵液，将发酵液分离纯化，获得AmB；所述发酵培养基成分为：葡萄糖60-80g/L，牛肉膏5-10g/L，大豆蛋白粉5-10g/L，棉子粉8-12g/L，CaCO35-10g/L，KH2PO4 0.1-0.4g/L，溶剂为自来水，pH 7.0，121度灭菌20min，优选：葡萄糖70g/L，牛肉膏8g/L，大豆蛋白粉8g/L，棉子粉10g/L，CaCO3 10g/L，KH2PO4 0.2g/L，溶剂为自来水，pH 7.0。
进一步，所述发酵培养在发酵罐中进行，发酵条件为25-30℃，发酵罐压力0.05MPa，搅拌转速200-500rpm(优选400rpm)，通气比0.08-1.5vvm(优选1.2vvm)。
进一步，所述产AmB的重组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％(优选5％)的接种量接种至发酵培养基，所述种子培养为：将产AmB的重组结节链霉菌接种至GYM平板，28℃培养7天，取颜色发灰黑色的孢子，使用棉花棒将表面孢子洗脱至无菌水中，将洗下的孢子悬液用含有棉花的注射器过滤，12000rpm离心5min后去上清液，沉淀加入无菌水重悬后，12000rpm离心5min重新洗脱一次，用无菌水重悬作为孢子悬液；所述GYM平板终浓度组成：葡萄糖4g/L，酵母粉4g/L，麦芽提取物10g/L，碳酸钙2g/L，琼脂18g/L，溶剂为自来水，pH7.2；将孢子悬液接种至种子培养基中，28℃，220rpm培养46h，获得种子液；所述种子液培养基终浓度组成为：蛋白胨10-20g/L，NaCl 5-10g/L，葡萄糖10-15g/L，酵母粉5-10g/L，CaCO3 0.5-1g/L，溶剂为自来水，pH 7.0，121度灭菌20min，优选蛋白胨20g/L，NaCl 8g/L，葡萄糖15g/L，酵母粉10g/L，CaCO3 1g/L，溶剂为自来水，pH 7.0。
本发明所述重组载体导入宿主菌方法为种属间接合转移方法：
1)将PCR克隆获得的卡那霉素抗性基因(Kan)及其启动子插入pJTU1278载体质粒多克隆位点处，得到重组载体pJTU1278-Kan；
2)将步骤1)中得到的重组载体转化入大肠杆菌JM109，对得到的转化子进行测序，将确认无误的载体导入供体菌大肠杆菌ET12567/pUZ8002；
3)将步骤2)中得到的含有重组载体的供体菌大肠杆菌ET12567/pUZ8002/pJTU1278-Kan，通过接合转移方法转化到受体菌中，得到具有卡那霉素(Kan)抗性的产AmB的基因工程菌。
本发明通过表达透明颤菌血红蛋白(vhb)和S-腺苷甲硫氨酸合成酶基因(metk)进一步提高AmB产量。
与现有技术相比，本发明有益效果主要体现在：
1、本发明通过导入卡那霉素抗性基因，使原本不具抗性的生产菌株带有卡那霉素抗性，提高种子培养基的菌种纯度和质量，实验室种子培养未出现染菌现象。
2、菌种带有抗生素抗性后，具有更低的染菌率，降低发酵时因染菌而失败的次数，降低对发酵环境的要求，并降低发酵过程中人为因素和环境因素的影响率。实验室5L罐发酵未出现染菌现象。
3、卡那霉素价格优廉、抗菌谱广、杀菌作用强，适用于工业中使用，对于企业总体收益率有较强提高作用，以实验室5L罐为计算，能降低成本约2000元/罐。
4、通过表达功能基因透明颤菌血红蛋白(vhb)，提高AmB产量约15％；通过表达功能基因S-腺苷甲硫氨酸合成酶基因(metk)，提高AmB产量约40％。
(四)附图说明
图1AmB结构式。
图2本发明实施例2构建的重组载体pJTU1278-Kan图谱；
图3实施例8中AmB高效液相色谱(HPLC)检测标准曲线。
图4实施例5中摇瓶发酵液中葡萄糖含量变化趋势图。
图5实施例5中摇瓶发酵过程pH变化趋势图。
图6实施例5中摇瓶发酵过程菌体干重变化趋势图。
图7实施例9中摇瓶发酵过程中不同发酵缓冲液体系影响。
图8实施例10中摇瓶发酵过程中不同pH对摇瓶发酵的影响。
图9实施例11中摇瓶发酵过程中不同温度对摇瓶发酵的影响。
图10实施例3中pJTU-vhb载体构建过程及重组载体图谱。
图11实施例12中重组结节链霉菌ZJB16050-Kan-vhb摇瓶发酵结果。
图12实施例4中pJTU-metk载体构建过程及重组载体图谱。
图13实施例13中重组结节链霉菌ZJB16050-Kan-metk摇瓶发酵结果。
(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：
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的试验材料，如无特殊说明，均为常规生化试剂。
实施例1、高产AmB突变株的筛选
(1)将结节链霉菌(Streptomyces nodosus，Caffrey P,Lynch S,Flood E,FinnanS,Oliynyk M.Amphotericin biosynthesis in Streptomyces nodosus:deductions fromanalysis of polyketide synthase and late genes.Chem Biol 2001；8:713-723.或Carmody M,Byrne B,Murphy B,Breen C,Lynch S et al.Analysis and manipulation ofamphotericin biosynthetic genes by means of modified phage KC515transductiontechniques.Gene 2004；343(1):107-115)接种至GYM平板或斜面培养基，28℃培养7天，取颜色发灰黑色的孢子，使用棉花棒将表面孢子洗脱至10mL无菌水中，将洗下的孢子悬液用含有棉花的注射器过滤，过滤后的孢子12000rpm离心5min后去上清液，加入10mL无菌水重悬后，12000rpm离心5min重新洗脱一次，用5mL无菌水重悬作为孢子悬液。
(2)EMS-UV诱变方法：将步骤(1)制备好的Streptomyces nodosus的孢子悬液5mL置于直径9cm无菌平皿中，在紫外灯下25cm处振荡照射30s，取一定量转于无菌试管，并立即浸入冰水中2h后，取样涂布于GYM平板中，28℃避光培养24-32h，收集诱变后菌体，使用EMS处理，处理方式：每1mL菌体悬液使用含5mg/mL甲基磺酸乙酯(EMS)的50mM、pH7.0的PBS缓冲液搅拌0.5h，8000rpm离心5min，收集菌体使用无菌水清洗3次，重悬后涂布在GYM固体培养基中，28℃避光培养，初步筛选颜色偏黄的菌株，挑取单菌落，按照实施例3进行发酵，收集发酵液通过HPLC方法检测产量(方法同实施例6)，直至获得AmB产量提升明显的突变株。突变次数、突变率和致死率如表1所示。
表1紫外-亚硝基胍复合诱变过程
(3)Co60诱变方法：将步骤(2)活化的菌株涂布于GYM固体培养基，28℃培养7天至长出孢子，收集孢子12000rpm离心5min，用无菌生理盐水洗涤3次，收集孢子，悬浮在生理盐水中，控制菌数约为108个/mL，以不同的剂量(0GY、200GY、400GY、600GY、800GY、1000GY和1200GY)的Co60对孢子进行诱变。将经Co60诱变的孢子用生理盐水稀释至10-5后涂板于GYM平板培养基，30℃培养5天，初步筛选颜色偏黄的菌株，挑取单菌落，按照实施例3进行发酵，进行后续的发酵培养，收集发酵液通过HPLC方法检测产量(方法同实施例6)，直至获得高产AmB突变株。突变次数、突变率和致死率如表2所示。
表2 Co60诱变方法诱变过程
(4)离子诱变方法：将步骤(3)活化的菌株涂布于GYM固体培养基，28℃培养7天至长出孢子，收集孢子12000rpm离心5min，用无菌生理盐水洗涤3次，收集孢子，悬浮在生理盐水中，控制菌数约为108个/mL，均匀涂在GYM固体培养基上，无菌风干。将带菌平板置IBBDevice 1多功能离子注入机靶室内，脉冲25，能量35keV，离子束流200mA，按2、8、20、40、60、80、100、200×1014ions·cm-2剂量进行照射。将上述经过照射的带菌平板以及未接受照射的对照带菌平板，用0.5mL无菌水洗脱，涂布到GYM平板培养基中，放到28℃的培养箱中培养24h，初步筛选颜色偏黄的菌株，挑取单菌落，按照实施例3进行发酵，收集发酵液通过HPLC方法检测AmB产量(方法同实施例6)，直至获得高产AmB突变株。突变次数、突变率和致死率如表3所示。
表3离子诱变过程
对于每轮诱变获得的高产菌株，重新作为初始菌株按照上述方法进行复合诱变。最终筛选获得AmB产量达14～16g/L的突变株ZJB2016050，命名为结节链霉菌(Streptomyces nodosus)ZJB2016050，保藏于中国典型培养物保藏中心，保藏日期2017年7月17号，保藏编号CCTCCNO：M 2017426，保藏地址为中国武汉，武汉大学，邮编430072。
本发明包含但不仅限于上述三种诱变方式。
其中GYM固体培养基的配制：葡萄糖4g/L，酵母粉4g/L，麦芽提取物10g/L，碳酸钙2g/L，琼脂18g/L，溶剂为自来水，pH7.2，121度灭菌20min。
实施例2、卡那霉素抗性的AmB基因工程菌构建
一、重组载体的构建
1、重组载体pJTU1278-Kan的构建
以pEC-XK99E质粒为模板(质粒购自BioVector NTCC质粒载体菌种细胞基因保藏中心，GenBank accession No.AY219682.1，Kirchner O,Tauch A.Tools for geneticengineering in the amino acid-producing bacterium Corynebacteriumglutamicum.J Biotechnol 2003；104(1-3):287-299.)，设计引物99E-Kan-F和99E-Kan-R，99E-Kan-F为针对Kan抗性相关基因的正向引物，99E-kan-R为针对Kan抗性相关基因的反向引物，从模板中克隆扩增出含卡那霉素抗性基因相关基因包括卡那霉素抗性基因(即aph(3')-IIa基因)、启动子以及RBS，片段大小1100bp左右，与目的片段相符，经测序分析，结果表明扩增得到的序列与NCBI上编号为AY219683.1的aph(3')-IIa基因序列相同，aph(3')-IIa基因的核苷酸序列如SEQ ID NO.1所示，该核苷酸序列所编码的卡那霉素抗性蛋白的氨基酸序列如SEQ ID NO.2所示。将此片段用核酸内切酶BamHI和HindIII酶切后，clean-up此片段备用，将载体pJTU1278也用同样的BamHI和HindIII核酸内切酶酶切胶回收，将回收后的基因片段与酶切后的pJTU1278载体连接，得到重组质粒载体命名为pJTU1278-Kan，示意图见图2。
其中克隆PCR体系：加入pEC-XK99E质粒模板1μL，加入2×Phanta Max Buffer 25μL，dNTP(2.5mM)5μL，99E-Kan正反引物各1μL，Phanta Max DNA聚合酶1μL，补足去离子水至50μL。
其中克隆PCR程序：98℃变性10s，55-60℃退火15s，72℃延伸1min，共30个循环。最后72℃延伸10min。
其中连接过程：将T4 DNA连接酶buffer 1μL加入灭菌的PCR管中，加入回收的DNA片段4μl和载体DNA 1μl，加入T4 DNA连接酶1μl，加入ddH2O 3μl，16℃反应20小时。将连接产物转化入JM109大肠杆菌感受态，利用氨苄青霉素抗性筛选，挑选转化子进行验证。
所用引物如下：
| 99E-Kan-F | CGCGGATCCAGCTTCACGCTGCCG |
| 99E-Kan-R | CCCAAGCTTCGAACCCCAGAGTCCCGC |
2、重组载体pJTU1278-Am的构建
以pSET-152质粒为模板(质粒购自武汉淼灵生物科技有限公司，产品货号M00727，GenBank accession No.AJ414670.1，Bierman M,Logan R,Brien KO,Seno ET,NagarajaRao R et al.Plasmid cloning vectors for the conjugal transfer of DNA fromEscherichia coli to Streptomyces spp.Gene 1992；116(1):43-49.)，设计引物152-F和152-R，152-F为针对安普霉素抗性相关基因的正向引物，152-R为针对安普霉素抗性相关基因的反向引物，从模板中克隆扩增出含安普霉素抗性基因相关基因包括安普霉素抗性基因、启动子以及RBS，片段大小776bp左右，与目的片段相符，经测序分析与安普抗性基因(aac(3)IV)一致，NCBI上基因编号为CP021858.1，核苷酸序列如SEQ ID NO.3所示，该核苷酸序列所编码的安普霉素抗性蛋白的氨基酸序列如SEQ ID NO.4所示。将此片段用核酸内切酶BamHI和HindIII酶切后，clean-up此片段备用，将载体pJTU1278也用同样的BamHI和HindIII核酸内切酶酶切胶回收，将回收后的基因片段与酶切后的pJTU1278载体连接，得到重组质粒载体命名为pJTU1278-Am。其中PCR程序同重组载体pJTU1278-Kan所述。
| 152-F | GCGGATCCAGCAGAGCGAGGTAT |
| 152-R | CCAAGCTTTCAGCCAATCGACTG |
3、重组载体pJTU1278-Amp的构建
以pTrc99a质粒为模板(质粒购自武汉淼灵生物科技有限公司，产品货号P0389，GenBank accession No.U13872.1)，设计引物99A-F和99A-R，99A-F为针对Amp抗性相关基因的正向引物，99A-R为针对Amp抗性相关基因的反向引物，从模板中克隆扩增出含氨苄青霉素抗性基因相关基因包括氨苄青霉素抗性基因、启动子以及RBS，片段大小858bp左右，与目的片段相符，NCBI上基因编号为LT727425.1，经测序分析与氨苄青霉素基因一致，核苷酸序列如SEQ ID NO.5所示，该核苷酸序列所编码的氨苄青霉素抗性蛋白的氨基酸序列如SEQID NO.6所示。将此片段用核酸内切酶BamHI和HindIII酶切后，clean-up此片段备用，将载体pJTU1278也用同样的BamHI和HindIII核酸内切酶酶切胶回收，将回收后的基因片段与酶切后的pJTU1278载体连接，得到重组质粒载体命名为pJTU1278-Amp。其中PCR程序同重组载体pJTU1278-Kan所述。
| 99A-F | GCGGATCCATGAGTATTCAACAT |
| 99A-R | CCAAGCTTCCAATGCTTAATCA |
本发明包含但不仅限于上述三种抗性基因。
二、重组载体pJTU1278-Kan接合转移转化受体菌结节链霉菌
A)含重组载体pJTU1278-Kan的E.coil ET12567/puz8002供体菌的准备：
将构建好的重组载体pJTU1278-Kan，导入E.coil ET12567/puz8002大肠杆菌感受态中，利用氨苄青霉素(Amp+，50μg/mL)、氯霉素(Cm+，50μg/mL)、卡那霉素(Kan+，50μg/mL)抗性筛选，挑取阳性转化子，通过M13上下游引物菌落PCR验证，结果证明重组载体pJTU1278-Kan成功转化入E.coil ET12567/puz8002。具体操作如下：
E.coil ET12567/puz8002大肠杆菌感受态制备方法如下：
从菌种的甘油冻存管中取E.coil ET12567/puz8002大肠杆菌菌液在LB平板上分区划线，37℃培养至单菌落长出。挑取平板上的单菌落转移到2～5mL的LB培养基中37℃，200rpm过夜培养。取过夜培养的菌液200μL加入到20mL的LB培养基中37℃，200rpm培养至OD600为0.4～0.7。培养好的菌液转移至预冷的50mL离心管中，冰上静置10min。离心4℃，2500×g，5min。弃上清液，加入4mL 0.1mol/L CaCl2，冰上重悬后静置10min。离心4℃，2500×g，5min。弃上清，加入2mL 0.1mol/L CaCl2(溶液中含终浓度为15％的甘油)，重悬沉淀，冰上静置30min，获得E.coil ET12567/puz8002大肠杆菌感受态细胞。分装100μL/tube，-80℃保藏。
含重组载体pJTU1278-Kan的E.coil ET12567/puz8002供体菌的制备：
取1支上述的E.coil ET12567/puz8002大肠杆菌感受态细胞，冰浴5min，加入5μL浓度为200ng/μL的pJTU1278-Kan载体质粒，冰浴30min，于42℃水浴，热激90s，放回冰浴1min，加入600μL LB液体培养基，37℃，200rpm，培养1h。吸取200μL，均匀涂布于Kan+(终浓度50μg/mL)、Cm+(终浓度50μg/mL)、Amp+(终浓度50μg/mL)抗性的LB固体平板，于37℃培养箱培养14h。至长出含重组载体pJTU1278-Kan的E.coil ET12567/puz8002单菌落。
M13验证PCR体系：挑取单菌落，加入20μL无菌水，沸水浴5～10min，12000rpm离心1min。取1μL上清液作为模板，加入10×pfu Buffer 1μL，dNTP(2.5mM)0.1μL，M13正反引物各0.1μL，pfu DNA聚合酶0.1μL，补足去离子水至10μL。
M13验证PCR程序：98℃变性10s，55～60℃退火15s，72℃延伸1min，共30个循环。最后72℃延伸10min。
M13引物如下：
| M13(-21)F | TGTAAAACGACGGCCAGT |
| M13R | CAGGAAACAGCTATGAC |
将已导入pJTU1278-Kan质粒的ET12567/puz8002大肠杆菌，划线分离单菌落，37℃培养，挑单菌落于装有5mL LB培养基试管，并同时加入Kan+(终浓度50μg/mL)、Cm+(终浓度50μg/mL)、Amp+(终浓度50μg/mL)抗生素，37℃培养14h。转接500μL于50mL LB摇瓶中，同时加入Kan+(终浓度50μg/mL)、Cm+(终浓度50μg/mL)、Amp+(终浓度50μg/mL)抗性，37℃培养至OD600为0.35。用50mL离心管将供体菌离心，4000rpm，5min，再用50mL LB培养基洗两次，用5mL LB培养基重悬，4℃保存备用。
其中LB培养基由以下方法制得：蛋白胨10g，酵母粉5g，氯化钠5g，自来水定容至1L，pH自然，121度灭菌20min。
B)受体菌Streptomyces nodosus的制备
将实施例1筛选的Streptomyces nodosus ZJB2016050(CCTCC M 2017426)接种在GYM平板或斜面培养物上，28℃生长10d，获得灰黑色的孢子，使用棉花棒将表面孢子洗脱至10mL 2×YT培养基中，将洗下的孢子悬液用含有棉花的注射器过滤，过滤后的孢子12000rpm，离心5min后去上清，加入10mL 2×YT培养基重悬，12000rpm离心5min重新洗脱一次，最后用500μL 2×YT培养基重悬。将重悬好的孢子在50℃热激15～20min后，常温下备用。
其中2×YT培养基由以下方法制得：蛋白胨16g，酵母粉10g，氯化钠5g，自来水定容至1L，pH自然，121度灭菌20min。
GYM固体培养基配制方法：葡萄糖4g，酵母粉4g，麦芽提取物10g，碳酸钙2g，琼脂18g，自来水定容至1L，pH7.2，121度灭菌20min。
C)供、受体菌接合过程：
将步骤B)500μL热激完的孢子悬液与步骤A)500μL供体大肠杆菌悬液混合重悬后，6000rpm离心2min，去掉800μL上清液，剩余上清将沉淀重悬涂布在含有10mM氯化镁的MS固体培养基平板上。28℃培养20h后，涂布1mL含0.5mg萘碇酸和0.5mg硫链丝菌素抗生素的水溶液，继续28℃培养10天，至出现转化子。
将转化子在含有终浓度50μg/mL萘碇酸和终浓度50μg/mL卡那霉抗性的GYM固体平板上连续纯化3次，直至获得单一菌落，使用16S RNA上下游引物(16S-8和16S-1541)以及M13上下游引物(M13(-21)F、M13R)对转化子菌落PCR验证后，测序对比分析，证实质粒(pJTU1278-Kan)已导入受体菌Streptomyces nodosus ZJB2016050中，最终获得产AmB的基因工程菌，即重组结节链霉菌ZJB2016050-Kan。
其中MS固体培养基由以下方法制得：黄豆粉20g，甘露醇20g，琼脂20g，自来水定容至1L，用氢氧化钠调至pH 7.2，121度灭菌20min。使用前加入终浓度10mM无菌氯化镁。
其中M13验证PCR操作如步骤A)所述。
其中16sRNA验证PCR体系：挑取单菌落，加入20μL无菌水，沸水浴30min，12000rpm离心1min。取3μL上清液作为模板，加入2×Phanta Max Buffer 5μL，dNTP(2.5mM)0.1μL，16S正反引物各0.1μL，Phanta Max DNA聚合酶0.1μL，补足去离子水至10μL。
其中16S RNA验证PCR程序：98℃变性10s，55～60℃退火15s，72℃延伸1min 30s，共30个循环。最后72℃延伸10min。
其中所用引物如下：
| 16S-8 | AGAGTTTGATCCTGGCTCAG |
| 16S-1541 | AAGGAGGTGATCCAGCCGCA |
| M13(-21)F | TGTAAAACGACGGCCAGT |
| M13R | CAGGAAACAGCTATGAC |
实施例3、携带vhb基因的结节链霉菌基因工程菌构建
以透明颤菌血红蛋白(vhb)基因为模板(GenBank accession No.JN418989.1)，设计引物vhbF和vhbR，vhbF为针对vhb基因的正向引物，vhbR为针对vhb的反向引物，从模板中克隆扩增出vhb，片段大小450bp左右，经测序分析确认，与目的片段相符，核苷酸序列如SEQID NO.7所示。将此片段用核酸内切酶BamHI和HindIII酶切后，clean-up此片段备用，将实施例2已构建的载体pJTU1278-Kan也用同样的BamHI和HindIII核酸内切酶酶切胶回收，将回收后的基因片段与酶切后的pJTU1278-Kan载体连接。得到重组质粒载体命名为pJTU1278-Kan-vhb，示意图见图10。
其中克隆PCR体系：加入pET28b-vhb质粒模板1μL，加入2×Phanta Max Buffer 25μL，dNTP(2.5mM)5μL，vhb正反引物各1μL，Phanta Max DNA聚合酶1μL，补足去离子水至50μL。
其中克隆PCR程序：98℃变性10s，55～60℃退火15s，72℃延伸30s，共30个循环。最后72℃延伸10min。
其中连接过程：将T4 DNA连接酶buffer 1μL加入灭菌的PCR管中，加入回收的DNA片段4μL和载体DNA 1μL，加入T4 DNA连接酶1μL，加入ddH2O 3μL，16℃反应20小时。将连接产物转化入大肠杆菌JM109感受态，利用氨苄青霉素抗性筛选，挑选转化子进行验证。
所用引物如下：
| vhbF | CCCAAGCTTATGCTGGACCAGCAGACC |
| vhbR | CGCGGATCCTCACTCGACCGCCTGG |
按照实施例2的接合转移方法，将构建的pJTU1278-Kan-vhb质粒导入Streptomyces nodosus ZJB2016050(CCTCC M 2017426)，获得重组结节链霉菌ZJB2016050-Kan-vhb。
实施例4、携带metk基因的结节链霉菌基因工程菌构建
以实施例1所述结节链霉菌(Streptomyces nodosus)基因组为模板，设计引物metkF和metkR，metkF为针对metk基因的正向引物(metk,GenBank accessionNo.CP009313.1)，metkR为针对metk的反向引物，从模板中克隆扩增出metk片段大小1424bp左右，经测序分析确认，与目的片段相符，核苷酸序列如SEQ ID NO.8所示。将此片段用核酸内切酶BamHI和HindIII酶切后，clean-up此片段备用，将已构建的载体pJTU1278-Kan也用同样的BamHI和HindIII核酸内切酶酶切胶回收，将回收后的基因片段与酶切后的pJTU1278-Kan载体连接。得到重组质粒载体命名为pJTU1278-Kan-metk，示意图见图12。
其中克隆PCR体系：加入基因组模板1μL，加入2×Phanta Max Buffer 25μL，dNTP(2.5mM)5μL，metk正反引物各1μL，Phanta Max DNA聚合酶1μL，补足去离子水至50μL。
其中克隆PCR程序：98℃变性10s，55～60℃退火15s，72℃延伸1min 30s，共30个循环。最后72℃延伸10min。
其中连接过程：将T4 DNA连接酶buffer 1μL加入灭菌的PCR管中，加入回收的DNA片段4μL和载体DNA 1μL，加入T4 DNA连接酶1μL，加入ddH2O 3μL，16℃反应20小时。将连接产物转化入JM109大肠杆菌感受态，利用氨苄青霉素抗性筛选，挑选转化子进行验证。
所用引物如下：
| metkF | CCCAAGCTTGTGTCCCGTCGCCTGTTCA |
| metkR | CGCGGATCCCTACAGCCCCACTGCCTT |
按照实施例2的接合转移方法，将构建的pJTU1278-Kan-metk质粒导入Streptomyces nodosus ZJB16050(CCTCC M 2017426)，获得重组结节链霉菌ZJB2016050-Kan-metk。
实施例5、摇瓶发酵产AmB
(1)孢子悬液的制备：将实施例2制备的产AmB的重组结节链霉菌ZJB16050-Kan接种至GYM平板，28℃培养7天，取颜色发灰黑色的孢子，使用棉花棒将表面孢子洗脱至10mL无菌水中，将洗下的孢子悬液用含有棉花的注射器过滤，过滤后的孢子12000rpm离心5min后去上清液，加入10mL无菌水重悬后，12000rpm离心5min重新洗脱一次，用5mL无菌水重悬作为孢子悬液。
(2)种子液的制备：
将步骤(1)孢子悬液接种至种子培养基中，28℃，220rpm培养46h，获得种子液。
种子培养基按以下方法制得：蛋白胨20g，NaCl 8g，葡萄糖15g，酵母粉10g，CaCO31g，加自来水定容至1L，pH 7.0，121度灭菌20min。
(3)发酵培养
500mL规格摇瓶装样100mL发酵培养基，发酵时按体积浓度2％接种种子液，28℃，220rpm发酵培养168h。发酵过程中，其发酵液中葡萄糖含量变化如图4，发酵液中pH变化如图5；基因工程菌干重变化如图6。
发酵培养基组成：葡萄糖70g/L，牛肉膏8g/L，大豆蛋白粉8g/L，棉子粉10g/L，CaCO3 10g/L，KH2PO4 0.2g/L，溶剂为自来水，pH 7.0，121度灭菌20min。
上述基因工程菌通过摇瓶发酵生产，按照实施例8方法检测，所得发酵液中AmB含量为5261.179mg/L。
实施例6、5L发酵罐发酵生产AmB
将实施例3制备的产AmB基因工程菌(重组结节链霉菌ZJB2016050-Kan-vhb)孢子悬液或斜面培养物接种种子培养基中，28℃，220rpm培养48h获得种子液。
发酵条件：5L发酵罐中装发酵液量3L，接种量按体积浓度5％接种种子液，28℃，压力0.05MPa、通气比1.2vvm，转速400rpm，发酵培养100h，获得发酵液。
上述基因工程菌通过5L罐发酵生产，按照实施例8方法检测，所得发酵液中AmB含量为8237.29mg/L。
种子培养基按以下方法制得：蛋白胨20g，NaCl 8g，葡萄糖15g，酵母粉10g，CaCO31g，加自来水定容至1L，pH 7.0，121度灭菌20min。
5L发酵罐发酵培养基组成：葡萄糖70g/L，牛肉膏8g/L，大豆蛋白粉8g/L，棉子粉10g/L，CaCO3 10g/L，KH2PO4 0.2g/L，溶剂为自来水，pH 7.0，121度灭菌20min。
同样条件下，以实施例1筛选的重组结节链霉菌ZJB2016050为对照进行发酵培养。
比较实施例3制备的产AmB基因工程菌(重组结节链霉菌ZJB2016050-Kan-vhb)与对照菌(重组结节链霉菌ZJB2016050)在5L罐发酵情况。至此发酵10批次，同样操作下，对照菌染菌次数4次，实施例3制备的基因工程菌染菌次数为0。
实施例7、AmB成品的制备
取实施例6方法获得的发酵液500L(AmB含量9.2g/L)。将发酵液经板框过滤得湿菌丝，烘干，得到7.5kg干菌重，把烘干的菌丝投入萃取罐中，加入70L甲醇，降温至4℃，用盐酸调至pH 3.0，稳定一小时，过滤得滤液。滤液进入结晶罐，加入纯化水10L，用碱液调pH至6.0，升温至25℃，保温一小时，结晶完成，静置分层。过滤得固体物(即AmB结晶粉)，不断加甲醇洗涤，除去杂质，最后烘干，粉碎，得AmB粗成品，粗成品通过实施例8所述液相色谱进行检测，产量为14～16g/L，产品纯度为93％以上。
实施例8、AmB的HPLC检测方法
取实施例6方法制备的发酵液，按发酵液：DMSO为1：9的体积比与DMSO混合，室温下萃取20～30分钟，12000rpm离心5min，取上清用0.45μm有机滤膜过膜后通过高效液相色谱(HPLC)检测。
检测方法：色谱柱为C18柱(150×4.6mm)，柱温25℃，流速1mL/min，进样量20μL，色谱保留时间30min，检测波长为405nm。AmB的出峰时间为26.9min。
其中流动相配制方法：1.1g EDTA-Na2和4.1g醋酸钠用蒸馏水定容至1L，取此溶液900mL与700mL乙腈、400mL甲醇混合，乙酸调至pH5.0；
AmB产量计算方法：从sigma公司购买AmB的标准品，用DMSO配制不同浓度(0mg/L、200mg/L、400mg/L、800mg/L、1000mg/L)的AmB标准溶液，分别将上述标准液用HPLC检测出峰面积，根据峰面积和AmB标准溶液的浓度计算出标准曲线为Y＝0.0123X+2539.9，R2＝0.999(其中Y为AmB的浓度，X为峰面积)。将未知浓度的AmB样品通过HPLC检测可以得到一个峰面积，带入上述标准曲线公式得出浓度，标准曲线结果如图3所示。
实施例9、不同发酵缓冲液体系影响
将实施例5发酵培养基中CaCO3分别替换为同摩尔量的Tris-HCl，Na2HPO4-NaH2PO4，Na2HPO4-Citric cid，Citric cid-Sodium citrate，Na2B4O7·10H2O，作为缓冲液(剂)，以蒸馏水为对照，初始pH调至7.0，其它操作同实施例5，按照实施例8检测方法检测发酵液中AmB，结果如图7所示。
发酵过程以CaCO3作为缓冲剂效果最佳，结节链霉菌菌丝干重和AmB产量相对于不加任何缓冲体系对照组均提升，其中菌丝干重提高约50％，AmB产量提高约100％。以Na2HPO4-Citric cid和Na2B4O7·10H2O为缓冲剂的发酵实验中，菌丝生长正常但不产AmB。
实施例10、摇瓶发酵体系pH优化
将实施例5发酵培养基pH分别调至6.0、6.5、7.0、7.5、8.0、8.5、9.0，其它操作同实施例5，按照实施例8检测方法检测发酵液AmB，结果如图8所示。
发酵过程的pH在6.5-7.0之间较佳，其中在pH 6.5条件下，菌丝干重和AmB产量最高，菌丝干重和AmB产量相对于不调节pH对照组(pH 7.2)分别提高8％和28％。在pH高于8.5的发酵过程中，AmB产量下降大于25％。
实施例11、摇瓶发酵体系发酵温度优化
将实施例5中发酵温度分别改为25℃、28℃、30℃、32℃、37℃，其它操作同实施例5，按照实施例8检测方法检测发酵液AmB，结果如图9所示。
发酵温度在28℃最佳，于144小时达到最高产量约5g/L。当温度超过30℃时，AmB产量于120小时提前下降。
实施例12、透明颤菌血红蛋白(vhb,GenBank accession No.JN418989.1)对结节链霉菌产两性霉素作用
将实施例3所得的携带vhb基因的重组结节链霉菌ZJB2016050-Kan-vhb，按照实施例5所述方法进行摇瓶发酵试验。同样条件下，以Streptomyces nodosus ZJB2016050(CCTCC M 2017426)作为对照。
结果导入vhb基因的工程菌对培养基里的葡萄糖成分利用率提高，菌丝生长较快，相对应的AmB产量相对于对照菌Streptomyces nodosus ZJB2016050(CCTCC M 2017426)提高约15％，如图11所示。
实施例13、S-腺苷甲硫氨酸合成酶(metk,GenBank accession No.CP009313.1)对结节链霉菌产两性霉素作用
将实施例4所得的携带metk基因的重组结节链霉菌ZJB2016050-Kan-metk，按照实施例5所述方法进行摇瓶发酵试验。同样条件下，以Streptomyces nodosus ZJB2016050(CCTCC M 2017426)作为对照。
结果导入metk基因的工程菌对培养基里的葡萄糖利用率无明显变化，菌丝生长状况与对照菌种treptomyces nodosus ZJB2016050(CCTCC M 2017426)无明显差异，但AmB的产量提高约40％，如图13所示。
序列表
<110> 浙江工业大学
<120> 一种产两性霉素B的重组结节链霉菌及其应用
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atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60
ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120
gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180
caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240
ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300
gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360
cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420
atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480
gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac 540
ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600
ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660
atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720
ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780
gacgagttct tctga 795
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Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala
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caccggacct tggagttgtc tctgacacat tctggcgcct gccaaatgta aagcgcagcg 300
cccatccatt tgcctttgcg gcagcggggc cacaggcaga gcagatcatc tctgatccat 360
tgcccctgcc acctcactcg cctgcaagcc cggtcgcccg tgtccatgaa ctcgatgggc 420
aggtacttct cctcggcgtg ggacacgatg ccaacacgac gctgcatctt gccgagttga 480
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tcctgcatcc gccagaggcg ggatgcgaag aatgcgatgc cgctcgccag tc 772
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Ile Gly
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atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60
gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120
cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180
gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240
cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300
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tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420
ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480
gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540
cctacagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600
tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660
tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 720
cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780
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tcactgatta agcattgg 858
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ggcgtcacca tcaccacgac cttctacaag aacctgttcg ccaagcaccc ggaggtccgc 120
ccgctgttcg acatgggccg ccaggagtcc ctggagcagc cgaaggccct ggcgatgacg 180
gtcctggcgg ccgcgcagaa catcgagaac ctgccggcca tcctgccggc ggtcaagaag 240
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gagctgctgg gcgcgatcaa ggaggtcctg ggcgacgccg ccaccgacga catcctggac 360
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gccgtggaga cgctgatcac caccggcctg gtgcatgtgg ccggcgaggt caccaccaag 180
gcctacgcgg acatcgccac gctggtgcgc aacaagatcc tcgagatcgg ttacgactcc 240
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ccggacatcg cccagggtgt ggacacggcg tacgagacgc gtgtcgaggg cgacgacgac 360
gagctggacc ggcagggcgc cggtgaccag ggcctgatgt tcggttatgc gacggacgag 420
acgccgaccc tgatgccgct gccgatcttc ctggcccacc ggctgtccaa gcggctgtcg 480
gacgtccgca agaacggcac gatcccctat cttcgcccgg acggaaagac ccaggtcacc 540
atcgagtacg acggcgacaa ggcggcccgt ctcgacacgg tggtggtctc ctcgcagcac 600
gccagcgaca tcgacctgga gtccctgctg gcccccgaca tccgcgagtt cgtggtggag 660
ccggagctga gggcgctgct ggacgacggc atcaagctgg agaccgacgg ctaccggctg 720
ctggtcaacc cgaccggccg tttcgagatc ggcggcccga tgggtgacgc gggtctgacc 780
ggtcggaaga tcatcatcga cacctacggc ggtatggccc ggcacggcgg cggcgccttc 840
tccggcaagg acccgtccaa ggtggaccgc agcgccgcct acgcgatgcg ctgggtggcc 900
aagaacgtgg tcgccgcggg actggccgcc cgctgcgagg tccaggtcgc ctacgccatc 960
ggcaaggccg agccggtggg cctgttcgtg gagaccttcg gcacggccaa ggtggacgcc 1020
gagaagatcg agcacgcgat cgccgaggtc ttcgacctcc gcccggccgc gatcatccgc 1080
gacctcgacc tgctgcgccc gatctactcc cagaccgccg cgtacggcca cttcggccgt 1140
gagctccccg acttcacctg ggagcgcacc gaccgggtgg acgcgctgcg caaggcagtg 1200
gggctgtagg gatcccccaa tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa 1260
gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta cccgcaggac 1320
atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct ccgagagctg 1380
catcaggtcg gagacgctgt cgaacttttc gatcagaaac ttct 1424
Claims (1)
1.一种产两性霉素B的重组结节链霉菌，其特征在于所述重组结节链霉菌是将SEQ IDNO.1所示卡那霉素抗性基因与SEQ ID NO.7或SEQ ID NO.8所示外源基因序列导入结节链霉菌(Streptomyces nodosus)ZJB2016050获得的；所述结节链霉菌(Streptomycesnodosus)ZJB2016050保藏于中国典型培养物保藏中心，保藏日期2017年7月17号，保藏编号CCTCC NO：M 2017426，保藏地址为中国武汉，武汉大学，邮编430072。
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| CN110305926B (zh) * | 2019-05-27 | 2021-02-26 | 浙江工业大学 | 一种基于两性霉素b代谢途径的发酵方法 |
| CN110343650B (zh) * | 2019-05-28 | 2020-12-29 | 浙江工业大学 | 一种产两性霉素b的重组结节链霉菌及其应用 |
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