CN117143199A - 氧磺酰化纽莫康定b0及其制备方法和应用 - Google Patents
氧磺酰化纽莫康定b0及其制备方法和应用 Download PDFInfo
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- CN117143199A CN117143199A CN202210568966.XA CN202210568966A CN117143199A CN 117143199 A CN117143199 A CN 117143199A CN 202210568966 A CN202210568966 A CN 202210568966A CN 117143199 A CN117143199 A CN 117143199A
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- oxysulfonylated
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Classifications
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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
- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- 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
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
- C12N9/0081—Cholesterol monooxygenase (cytochrome P 450scc)(1.14.15.6)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/13—Transferases (2.) transferring sulfur containing groups (2.8)
-
- 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
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
- C12Y114/15006—Cholesterol monooxygenase (side-chain-cleaving) (1.14.15.6), i.e. cytochrome P450scc
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y208/00—Transferases transferring sulfur-containing groups (2.8)
- C12Y208/02—Sulfotransferases (2.8.2)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
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Abstract
本发明公开了一种氧磺酰化的纽莫康定B0，所述氧磺酰化纽莫康定B0的结构式如式(I)所示；本发明还提供了一种纽莫康定产生菌的基因工程菌株，所述工程菌株为在纽莫康定产生菌中引入细胞色素P450单加氧酶和/或磺酰基转移酶所得到的工程菌株；本发明还提供了细胞色素P450单加氧酶和/或磺酰基转移酶在制备氧磺酰化的纽莫康定B0中的用途；另外，本发明还提供了上述氧磺酰化的纽莫康定B0在制备抗真菌药物中的用途；本发明的氧磺酰化的纽莫康定B0与纽莫康定B0相比，其水溶性能够显著提升，与纽莫康定B0相比提高了4000多倍。
Description
技术领域
本发明属于生物制药技术领域，涉及一种氧磺酰化纽莫康定B0及其制备方法和应用。
背景技术
棘白菌素类药物是一种新型的抗真菌药物，通过非竞争性地抑制真菌细胞壁中β-1,3-葡聚糖合成酶的活性从而使真菌细胞壁无法合成，导致真菌细胞裂解死亡。
目前，临床上应用的棘白菌素类抗真菌药物有卡泊芬净(Caspofungin)、米卡芬净(Micafungin)和阿尼芬净(Anidulafungin)，主要用于治疗由念珠菌和曲霉等引起的深部真菌感染。其中卡泊芬净是由美国FDA批准上市的第一个棘白菌素类抗真菌药物，也是目前临床上使用最多的棘白菌素类抗真菌药物。卡泊芬净的前体是由Glarea lozoyensis产生的纽莫康定B0(Pneumocandins B0)。由于纽莫康定B0的水溶性较低，在卡泊芬净药物工业生产过程中，需要对纽莫康定B0经过两步化学修饰得到，化学修饰改善水溶性是重要关注点之一。对前体的化学修饰使得生产工艺更加复杂，也增加了生产成本。
本申请提供了一种水溶性更好的氧磺酰化的纽莫康定B0，在实际应用时具有更广阔的的前景。
发明内容
一方面，本发明提供了一种氧磺酰化的纽莫康定B0，所述氧磺酰化纽莫康定B0的结构式如式(I)所示：
另一方面，本发明还提供了上述氧磺酰化的纽莫康定B0在制备抗真菌的药物中的用途。
在一个实施方式中，所述真菌选自白色念珠菌、地方性真菌、曲霉及卡氏肺囊虫中的一种或任意几种。
另一方面，本发明还提供了一种纽莫康定产生菌(G.lozoyensis)的基因工程菌株，所述工程菌株为在纽莫康定产生菌(G.lozoyensis)中引入细胞色素P450单加氧酶和/或磺酰基转移酶所得到的工程菌株。
在一个实施方式中，所述纽莫康定产生菌(G.lozoyensis)为G.lozoyensis ATCC74030)。
在一个实施方式中，所述细胞色素P450单加氧酶与SEQ ID No.2相比具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述细胞色素P450单加氧酶来源于鞘茎点霉属真菌(Coleophoma sp.)；更优选的，所述细胞色素P450单加氧酶的氨基酸序列与SEQ ID No.2相比具有至少70％的序列同一性，并且所述细胞色素P450单加氧酶来源于鞘茎点霉属真菌；所述鞘茎点霉属真菌包括Coleophoma sp.或Coleophoma empetri，例如，鞘茎点霉(Coleophoma sp.)MEFC009。在其他的实施方式中，所述C.empetri为C.empetri F-11899。更优选的，所述细胞色素P450单加氧酶的氨基酸序列如SEQ ID No.2所示。
在一个实施方式中，所述磺酰基转移酶与SEQ ID No.4相比具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％的序列同一性；优选的，所述磺基转移酶来源于鞘茎点霉属真菌(Coleophoma sp.)；更优选的，所述磺基转移酶的氨基酸序列与SEQ ID No.4相比具有至少70％的序列同一性，并且所述磺基转移酶来源于鞘茎点霉属真菌；所述鞘茎点霉属真菌包括Coleophoma sp.或C.empetri，例如，鞘茎点霉(Coleophoma sp.)MEFC009。在其他的实施方式中，所述C.empetri为C.empetri F-11899。更优选的，所述磺基转移酶的氨基酸序列如SEQ ID No.4所示。
本发明中，鞘茎点霉(Coleophoma sp.)MEFC009，保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号为CGMCC No.21058，保藏日期为2020年11月18日，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，电话：010-64807355。
另一方面，本发明还提供了上述基因工程菌株在生产氧磺酰化纽莫康定B0中的应用。
所述氧磺酰化纽莫康定B0的结构式如式(I)所示。
另一方面，本发明还提供了一种制备氧磺酰化纽莫康定B0的方法，所述方法包括利用上述基因工程菌株进行发酵的步骤；进一步的，所述方法还包括分离或纯化所述氧磺酰化纽莫康定B0的步骤。
另一方面，本发明还提供了包含上述细胞色素P450单加氧酶或磺酰基转移酶或其编码基因的生物材料。所述生物材料选自：包含上述细胞色素P450单加氧酶或磺酰基转移酶的载体，或者，包含上述细胞色素P450单加氧酶或磺酰基转移酶的宿主细胞。
另一方面，本发明还提供了编码上述细胞色素P450单加氧酶或磺酰基转移酶的基因。
另一方面，本发明还提供了包含上述基因的载体，或者，包含所述载体的宿主细胞。
在一个实施方式中，所述载体包括克隆载体和表达载体，例如，pET系列载体(如pET-14、pET-21、pET-22、pET-28、pET-30、pET-42、pET-GST、pET-His、pET-Trx、pET-GST、pET-CKS、pET-DsbA)、pMAL系列载体(如pMAL-2c)、pGEX系列载体(如pGEX-4T-2、pGEX-6T-1)、pBAD系列载体(如pBAD-His、pBAD-Myc)、pMBP系列载体(pMBP-P、pMBP-C)、pTYB2、pQE-9、pACYCDuet-1、pCDFDuet-1、pColADuet-1、pRSFDuet-1、pllP-OmpA、pUC系列载体(如pUC18、pUC19)、pQE-30、pXH2-1、pXH-43、pTRII、pGSF957。
在一个实施方式中，所述宿主细胞选自大肠杆菌(例如，大肠杆菌DH5α、大肠杆菌BL21(DE3)、Rosetta(DE3)、Codon Plus(DE3)-RIPL、BL21 Codon plus(DE3)、Top10、JM109)、酵母菌(例如，酿酒酵母、毕赤酵母、解酯耶氏酵母)、鞘茎点霉真菌、纽莫康定产生菌(G.lozoyensis)。
另一方面，本发明还提供了上述细胞色素P450单加氧酶或磺酰基转移酶、其编码基因、包含基因的载体、上述宿主细胞、或上述生物材料在制备氧磺酰化的纽莫康定B0中的应用。
在一个实施方式中，本发明提供了上述细胞色素P450单加氧酶和磺酰基转移酶在催化纽莫康定B0形成氧磺酰化纽莫康定B0中的用途。
本发明中，所述细胞色素P450单加氧酶又称为P450酶。
所述的“引入”包括将上述目的基因在出发菌株中进行表达的步骤，优选，过表达。例如，将目的基因构建到表达载体上，将表达载体转入到宿主细胞中以表达目的基因，优选，过表达。在其他的实施方式中，所述的“引入”包括将目的基因插入到宿主细胞的基因组中；优选的，所述插入到宿主细胞的基因组中可以采用同源重组双交换的方法；在一个实施方式中，可以采用将目的基因以及同源臂插入到载体上，然后将载体转入到宿主细胞中，利用同源臂与宿主细胞基因组发生同源重组双交换从而将目的基因插入到合适的基因组的位置；在其他的实施方式中，还可以采用基因编辑的方式，例如，利用CRISPR/Cas系统在期望的基因组位点上进行切割，同时将目的基因作为外源供体插入到切割位点上。
本发明中的“过表达”是指基因工程菌与野生型的出发菌株相比，所述目的基因的表达量或活性要高于野生型的出发菌株。在一个实施方式中，上述过表达可以通过引入表达载体来过表达目的基因来实现；其他的实施方式中，上述过表达也可以通过在出发菌株中引入额外的目的基因的拷贝、通过增加目的基因的拷贝数来实现；其他的实施方式中，还可以通过对目的基因的启动子优化来实现，比如，通过将目的基因的原始启动子替换为启动子活性更高的启动子来实现目的基因的过表达。
另一方面，本发明还提供了上述基因工程菌的构建方法。
基于上述内容，本发明至少提供了以下技术方案：
一种氧磺酰化的纽莫康定B0，所述氧磺酰化纽莫康定B0的结构式如式(I)所示：
一种纽莫康定产生菌(G.lozoyensis)的基因工程菌株，所述工程菌株为在纽莫康定产生菌中引入细胞色素P450单加氧酶和磺酰基转移酶所得到的工程菌株；
所述细胞色素P450单加氧酶与SEQ ID No.2相比具有至少70％的序列同一性；
所述磺酰基转移酶与SEQ ID No.4相比具有至少70％的序列同一性。
一种制备氧磺酰化的纽莫康定B0的方法，所述方法包括利用上述基因工程菌株进行发酵的步骤。
上述细胞色素P450单加氧酶和磺酰基转移酶，或其编码基因在制备生产所述的氧磺酰化的纽莫康定B0的基因工程菌株中的应用，所述基因工程菌株的出发菌株为纽莫康定B0产生菌(G.lozoyensis ATCC 74030)。
上述细胞色素P450单加氧酶和磺酰基转移酶，或者包含所述的细胞色素P450单加氧酶和磺酰基转移酶的生物材料，或者上述基因工程菌株在制备氧磺酰化的纽莫康定B0中的应用。
所述生物材料选自：包含所述的细胞色素P450单加氧酶和磺酰基转移酶，或其编码基因的载体；或者，包含所述的细胞色素P450单加氧酶和磺酰基转移酶，或其编码基因的宿主细胞。
上述细胞色素P450单加氧酶和磺酰基转移酶在催化纽莫康定B0形成所述的氧磺酰化的纽莫康定B0中的应用。
一种制备所述的氧磺酰化的纽莫康定B0的方法，所述方法包括利用所述的细胞色素P450单加氧酶和磺酰基转移酶催化纽莫康定B0形成所述氧磺酰化的纽莫康定B0中的步骤。
所述的氧磺酰化的纽莫康定B0，或者上述方法制备得到的氧磺酰化的纽莫康定B0在制备抗真菌药物中的用途。
一种抗真菌药物，所述药物包括所述的氧磺酰化的纽莫康定B0，或者上述所述方法制备得到的氧磺酰化的纽莫康定B0。
附图说明
图1为敲除mcfP基因所获转化子的基因组PCR验证结果；其中6#，8#，9#是基因mcfP缺失的转化子，WT-1为对照菌株Coleophoma sp.-Δku80。
图2为基因mcfP缺失菌株Coleophoma sp.-ΔmcfP发酵产物HPLC分析结果；其中Coleophoma sp.-ΔmcfP为基因mcfP缺失菌株，WT-1为Coleophoma sp.-Δku80。
图3为化合物4，5，6，7和8的LC-MS分析结果，其中A为化合物4，B为化合物5，C为化合物6，D为化合物7，E为化合物8。
图4为化合物4，5，6，7和8的结构。
图5为敲除mcfS基因所获转化子的基因组PCR验证结果；其中1#，3#，7#是基因mcfS缺失的转化子，WT-1为对照菌株Coleophoma sp.-Δku80。
图6为基因mcfS缺失菌株Coleophoma sp.-ΔmcfS发酵产物HPLC分析结果；其中Coleophoma sp.-ΔmcfS为基因mcfS缺失菌株，WT-1为Coleophoma sp.-Δku80。
图7为化合物9的LC-MS分析结果。
图8为重组质粒pCAMBIA1300-mcfP和pCAMBIA1300-mcfS图谱示意图；其中A为质粒pCAMBIA1300-mcfP，B为pCAMBIA1300-mcfS。
图9为异源表达P450酶McfP和磺基转移酶McfS的G.lozoyensis ATCC 74030转化子的基因组PCR验证结果；其中1-9为转化子；WT-2为对照菌株G.lozoyensis ATCC。
图10为工程菌株G.lozoyensis::mcfP::mcfS发酵产物HPLC分析结果；其中，G.lozoyensis::mcfP::mcfS为同时异源表达基因mcfP和mcfS的重组菌株，G.lozoyensisATCC 74030为对照菌株。
图11为化合物11和12的LC-MS分析结果，其中A为化合物11，B为化合物12。
图12为化合物11和12的化学结构。
图13为异源表达P450酶McfP的G.lozoyensis ATCC 74030转化子的基因组PCR验证结果；其中1-18为转化子；WT-2为对照菌株G.lozoyensis ATCC 74030。
图14为工程菌株G.lozoyensis::mcfP发酵产物HPLC分析结果；其中，G.lozoyensis::mcfP为异源插入基因mcfP的重组菌株，G.lozoyensis ATCC 74030为对照菌株。
图15为纽莫康定B0、化合物11和化合物12在水中的溶解度。
图16为纽莫康定B0、化合物11和化合物12对白色念珠菌的抑菌活性分析。1：两性霉素B阳性对照，2：纽莫康定B0，3：化合物11；4：化合物12；5：DMSO阴性对照。
具体实施方式
下面结合具体实施例对本发明做进一步说明，但本发明不受实施例的限制。以下实施例中所用材料、试剂、仪器和方法，未经特殊说明，均为本领域中的常规材料、试剂、仪器和方法，均可通过商业渠道获得。
本发明中质粒提取采用OMEGA公司的Plasmid Mini Kit I试剂(D6942-01)；PCR片段纯化采用OMEGA公司DNA片段回收Cycle-Pure Kit试剂盒(D6492-01)；一步克隆酶Ultra One Step Cloning Kit购自南京Vazyme公司；限制性内切酶购自Thermo公司；T4连接酶购自New England Biolabs；RNA提取采用TAKARA公司的Mini BESTPlant RNA Extraction Kit试剂盒；cNDA反转录试剂盒购自TAKARA公司；大肠杆菌感受态细胞DH5α和BL21(DE3)购自南京Vazyme公司；农杆菌感受态细胞LBA4404购自唯地生物公司。
大肠杆菌培养基LB培养基：1％蛋白胨，0.5％酵母粉，1％NaCl，pH 7.0。
Coleophoma sp.的种子培养基：15g/L可溶性淀粉，10g/L蔗糖，5g/L棉籽饼粉，10g/L蛋白胨，1g/L KH2PO4，2g/L CaCO3，pH 6.0-8.0。
Coleophoma sp.的发酵培养基：30g/L玉米淀粉，30g/L蛋白胨，6g/L(NH4)2SO4，1g/L KH2PO4，0.3g/L FeSO4·7H2O，0.01g/L ZnSO4·7H2O，2g/L CaCO3，pH 6.0-8.0。
G.lozoyensis ATCC 74030的种子培养基：20g/L大豆粉，40g/L葡萄糖，1g/LKH2PO4，pH 5.0-8.0。
G.lozoyensis ATCC 74030的发酵培养基：100g/L甘露醇，20g/L葡萄糖，10g/L棉籽饼粉，10g/L蛋白胨，2.5g/L K2HPO4·3H2O，pH 5.0-8.0。
STC：1M山梨醇，50mM Tris-HCl(pH＝8.0)，50mM CaCl2。
PSTC：40％PEG4000，1M山梨醇，50mM Tris-HCl(pH＝8.0)，50mM CaCl2。
顶层琼脂：PDB、1M山梨醇和4g/L琼脂糖，灭菌后48℃保温。
再生筛选培养基平板PDA-SH：PDA平板、1M山梨醇和100mg/L潮霉素B。
筛选培养基PDA-H：PDA平板和100mg/L潮霉素B。
质粒pXH2-1记载在Xuenian Huang,Xuefeng Lu,Jian-Jun Li.Cloning,characterization and application of a glyceraldehyde-3-phosphatedehydrogenase promoter from Aspergillus terreus,J Ind Microbiol Biotechnol(2014)41:585-592。
质粒pCAMBIAMBIA1300记载在Hana Martina Hujslová,MilanGryndler.Genetic transformation of extremophilic fungi Acidea extrema andAcidothrix acidophila,Folia Microbiol(Praha).2015,60(4),365-71.
质粒pPM-3记载在Ping Men,Min Wang,Jinda Li,Xuenian Huang,XuefengLu.Establishing an efficient genetic manipulation system for sulfatedechinocandin producing fungus Coleophoma empetri.Frontiers inMicrobiology.2021,12,734780。
质粒pCAMBIA1300-mcfP(本实验室自主构建)
质粒pCAMBIA1300-mcfS(本实验室自主构建)
鞘茎点霉属真菌(Coleophoma sp.)保藏于中国普通微生物菌种保藏管理中心，(该菌株保藏号是CGMCC NO:21058，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)。
Glarea lozoyensis ATCC 74030购自American type culture collection。
白色念珠菌Canidia albicans[CMCC(F)98001]购自中国普通微生物菌种保藏管理中心。
实施例1.构建敲除mcfP基因的工程菌株Coleophoma sp.-ΔmcfP
以野生型Coleophoma sp.MEFC009的基因组为模板，采用pfu DNA聚合酶(Fermentas,Catalog No.:EP0501)进行PCR扩增，用引物UmcfP-F(5’-tctcaaggagataactcccacac-3’)和UmcfP-R(5’-ctttacgcttgcgatcccgaaTCATTGGGATTGATGCGGATGATAGG-3’)可以扩增获得大小约为1.2kb的mcfP的上游序列U-mcfP，用引物DmcfP-F(5’-ccctgggttcgcaaagataattgCGTATCTTTCCACTAATACTGC-3’)和DmcfP-R(5’-caccgtacctgaatcctcat-3’)可以扩增获得大小为1.2kb的mcfP的下游序列D-mcfP。以质粒pXH2-1为模板，用引物hph-F(5’-ttcgggatcgcaagcgtaaag-3’)和hph-R(5’-caattatctttgcgaacccagg-3’)进行PCR扩增，获得大小约为2.2kb的潮霉素抗性筛选片段hph；通过融合PCR将hph片段、上游序列U-mcfP和下游序列D-mcfP进行融合，再以融合产物为模板，用巢式引物UmcfP-CS-F(5’-ggacaacgaatagctaaatgaaga-3’)和DmcfP-CS-R(5’-gctctgctattcataactcg-3’)通过PCR扩增出大小为4.4kb的敲除打靶元件UmcfP-hph-DmcfP。所述mcfP基因序列如SEQ ID No.1所示，所述McfP的氨基酸序列如SEQ ID No.2所示。
以Coleophoma sp.-Δku80为出发菌，首先从PDA平板上取少量菌丝，用手持匀浆器破碎，取1mL种子液接种到50mL的种子培养基，于250mL的三角瓶中，220rpm，25℃进行摇床培养。2天后，离心收集菌丝。5000rpm，4℃，5min。将菌丝体再次用匀浆器破碎，取0.5mL-2mL种子液接种到50mL的种子培养基，相同的条件下培养1天，将培养基与菌丝体一起倒入50mL的无菌离心管中，5000rpm，离心收集菌丝。用0.6M MgSO4对菌丝体进行清洗2次。将菌丝体清洗至白色，称取1g菌丝，加入10mL酶解液，30℃，100rpm处理1-4h。酶解液成分为：1％纤维素酶、0.6％溶壁酶、0.6％蜗牛酶、0.6M MgSO4，经由0.22μm的无菌过滤器过滤除菌。将上述原生质体反应液通过无菌神奇滤布进行过滤。5000rpm，4℃离心收集原生质体。用冰预冷的STC，洗涤一次，把原生质体重悬于预冷的STC中，并用STC将原生质体浓度调整为5×107个/mL，得到原生质体悬液。
向140μL上述原生质体悬液中加入10μL的UmcfP-hph-DmcfP片段，再加入50μL的PSTC，轻轻混匀，冰浴30min。加入1mL PSTC，混匀后室温放置20min；然后与10mL顶层琼脂混合后倾注于3块再生筛选培养基平板PDA-SH上，在30℃、黑暗条件下培养5-7天，得到转化子。
从转化筛选平板上挑取具有潮霉素抗性转化子转移至PDA-H上，在25℃培养4-6天进行传代培养，连续传代3代。选取稳定传代的3个转化子(6#，8#，9#)进行单孢分离纯化，并提取单孢分离之后的转化子基因组。利用外部引物UmcfP-F(5’-tctcaaggagataactcccacac-3’)和DmcfP-R(5’-caccgtacctgaatcctcat-3’)对转化子基因组进行PCR验证，能扩增出大小约为4.6kb的条带的为阳性转化子，而Coleophoma sp.-Δku80只能扩增出大小约为2.9kb的条带，图1说明6#，8#，9#转化子为阳性转化子，说明在基因mcfP位置发生了同源重组，整合了外源片段UmcfP-hph-DmcfP。
实施例2.mcfP基因缺失工程菌株Coleophoma sp.-ΔmcfP的发酵及产物分析
将3株mcfP基因缺失工程菌株Coleophoma sp.-ΔmcfP 6#，8#，9#和对照菌株Coleophoma sp.-Δku80接种于PDA固体平板上，25℃培养4-6天。挑取少量的菌丝，利用核酸提取仪(-24)对菌丝进行破碎，将破碎后的菌丝接种于50mL Coleophoma sp的种子培养基(250mL三角瓶)，25℃，220rpm，摇床培养48h。将上述培养的种子液取5mL到Coleophoma sp.的发酵培养基，25℃，220rpm，摇床培养8天，每个菌株设置3个平行。从每一瓶发酵液中取1mL，加入等体积的甲醇，超声萃取1h，离心，取上清。用0.22μm的有机滤器过滤处理样品，并通过HPLC和LC-MS对处理后的样品进行分析。
HPLC分析方法为：液相色谱柱为Agilent C-18反向柱883975-902(4.6×150mm，5μm)；流动相为A：0.05％(体积比)三氟乙酸水溶液，流动相B：0.05％(体积比)三氟乙酸乙腈溶液，流速为1mL/min，紫外检测波长：210nm，30℃，总洗脱时间为37min。梯度洗脱条件：0-5min，流动相B占流动相的体积由5％线性上升到24％，5-35min，流动相B占流动相的体积由24％线性上升到62％，35-37min，流动相B占流动相的体积由62％线性上升到100％。结果如图2所示；与出发菌株Coleophoma sp.-Δku80相比，化合物1、2、3消失，相应的出现了紫外吸收与化合物1、2、3相同的另外4个化合物。进而对化合物4、5、6、7和8进行分离纯化，通过液质联用(LC-MS)和核磁共振(NMR)进行分析。LC-MS分析方法为：Agilent公司1290高效液相色谱，色谱柱为Agilent Zorbax Extend-C18的C18柱(2.1×50mm，1.8μm)；流动相总流速为0.6mL/min；流动相A：0.05％(体积比)甲酸水溶液，流动相B：0.05％(体积比)甲酸乙腈溶液，总洗脱时间为7.0min；洗脱条件为：梯度洗脱条件：0-1min，流动相B占流动相的体积由5％线性上升到20％，1-6min，流动相B占流动相的体积由20％线性上升到60％，6-7min，流动相B占流动相的体积由60％线性上升到100％。结果如图3所示；结合NMR分析结果可知，4(分子式：C51H82N8O17，理论值：[M+H]+1079.5871，实际值：1079.5873),5(分子式：C50H80N8O16，理论值：[M+H]+1049.5765，实际值：1049.5766)，6(分子式：C51H82N8O16，理论值：[M+H]+1063.5922，实际值1063.5921),7(分子式：C51H82N8O15，理论值：[M+H]+1047.5972，实际值：1047.5969)和8(分子式：C51H82N8O14，理论值：[M+H]+1031.6023，实际值：1031.6022)，根据分子量推测这些中间体少了氧磺酰基和一些羟基。进一步通过NMR对化合物4、5、6、7和8的结构进行了鉴定。他们有一个共同的特征：氧磺酰基消失，进而说明基因mcfP编码的P450酶负责FR901379结构中氧磺酰化模块形成的第一步——高酪氨酸苯环C3位的羟基化。
以McfP的氨基酸序列作为参照，在另外3株产其他磺酰化的棘白菌素类化合物生产菌株Coleophoma cylindrospora，Coleophoma crateriform和Venustampullaechinocandica BP5553中找到了同源性大于75％的同源序列，在NCBI数据库中的序列号分别为RDW63434.1、RDW57263.1和XP_031866084.1。这3个蛋白很有可能与McfP具有相同的功能，负责氧磺酰基模块中第一步羟基的形成。
实施例3.构建敲除mcfS基因的工程菌株Coleophoma sp.-ΔmcfS
以野生型Coleophoma sp.的基因组为模板，采用pfu DNA聚合酶(Fermentas,Catalog No.:EP0501)进行PCR扩增，用引物UmcfS-F(5’-gcgccttcgaagcgggcaac-3’)和UmcfS-R(5’-ctttacgcttgcgatcccgaaTCGAAGGCCTCTTTCCACAAC-3’)可以扩增获得大小约为1.2kb的mcfS的上游序列U-mcfS，用引物DmcfS-F(5’-cctgggttcgcaaagataattgACATATTCAAGTACAGCCCCC-3’)和DmcfS-R(5’-tagtccagaggatgacttcc-3’)可以扩增获得大小为1.2kb的mcfS的下游序列D-mcfS。以质粒pXH2-1为模板，用引物hph-F(5’-ttcgggatcgcaagcgtaaag-3’)和hph-R(5’-caattatctttgcgaacccagg-3’)进行PCR扩增，获得大小约为2.2kb的潮霉素抗性筛选片段hph；通过融合PCR将hph片段、上游序列U-mcfS和下游序列D-mcfS进行融合，再以融合产物为模板，用巢式引物UmcfS-CS-F(5’-gaatactttgctcgcaggtg-3’)和DmcfS-CS-R(5’-gccaatctataaagggaaagg-3’)通过PCR扩增出大小为4.4kb的敲除打靶元件UmcfS-hph-DmcfS。
以Coleophoma sp.-Δku80为出发菌，首先从PDA平板上取少量菌丝，用手持匀浆器破碎，取1mL种子液接种到50mL的种子培养基，于250mL的三角瓶中，220rpm，25℃进行摇床培养。2天后，离心收集菌丝。5000rpm，4℃，5min。将菌丝体再次用匀浆器破碎，取0.5mL-2mL种子液接种到50mL的种子培养基，相同的条件下培养1天，将培养基与菌丝体一起倒入50mL的无菌离心管中，5000rpm，离心收集菌丝。用0.6M MgSO4对菌丝体进行清洗2次。将菌丝体清洗至白色，称取1g菌丝，加入10mL酶解液，30℃，100rpm处理1-4h。酶解液成分为：1％纤维素酶、0.6％溶壁酶、0.6％蜗牛酶、0.6M MgSO4，经由0.22μm的无菌过滤器过滤除菌。将上述原生质体反应液通过无菌神奇滤布进行过滤。5000rpm，4℃离心收集原生质体。用冰预冷的STC，洗涤一次，把原生质体重悬于预冷的STC中，并用STC将原生质体浓度调整为5×107个/mL，得到原生质体悬液。
向140μL上述原生质体悬液中加入10μL的UmcfS-hph-DmcfS片段，再加入50μL的PSTC，轻轻混匀，冰浴30min。加入1mL PSTC，混匀后室温放置20min；然后与10mL顶层琼脂混合后倾注于3块再生筛选培养基平板PDA-SH上，在30℃黑暗条件下培养5-7天，得到转化子。
从转化筛选平板上挑取具有潮霉素抗性转化子转移至PDA-H上，在25℃培养4-6天进行传代培养，连续传代3代。选取稳定传代的3个转化子(1#，3#，7#)进行单孢分离纯化，并提取单孢分离之后的转化子基因组。利用外部引物UmcfS-F(5’-gcgccttcgaagcgggcaac-3’)和DmcfS-R(5’-tagtccagaggatgacttcc-3’)对转化子基因组进行PCR验证，能扩增出大小约为4.9kb的条带的为阳性转化子，而Coleophoma sp.-Δku80只能扩增出大小约为3.1kb的条带，图5说明1#，3#，7#转化子为阳性转化子，说明在基因mcfS位置发生了同源重组，整合了外源片段UmcfS-hph-DmcfS。
实施例4.mcfS基因缺失工程菌株Coleophoma sp.-ΔmcfS的发酵及产物分析
将3株mcfS基因缺失工程菌株Coleophoma sp.-ΔmcfS 1#，3#，7#和对照菌株Coleophoma sp.-Δku80接种于PDA固体平板上，25℃培养4-6天。挑取少量的菌丝，利用核酸提取仪(-24)对菌丝进行破碎，将破碎后的菌丝接种于50mL Coleophoma sp.的种子培养基(250mL三角瓶)，25℃，220rpm，摇床培养48h。将上述培养的种子液取5mL到Coleophoma sp的发酵培养基，25℃，220rpm，摇床培养8天，每个菌株设置3个平行。从每一瓶发酵液中取1mL，加入等体积的甲醇，超声萃取1h，离心，取上清。用0.22μm的有机滤器过滤处理样品，并通过HPLC和LC-MS对处理后的样品进行分析。
HPLC分析方法为：液相色谱柱为Agilent C-18反向柱883975-902(4.6×150mm，5μm)；流动相为A：0.05％(体积比)三氟乙酸水溶液，流动相B：0.05％(体积比)三氟乙酸乙腈溶液，流速为1mL/min，紫外检测波长：210nm，30℃，总洗脱时间为37min。梯度洗脱条件：0-5min，流动相B占流动相的体积由5％线性上升到24％，5-35min，流动相B占流动相的体积由24％线性上升到62％，35-37min，流动相B占流动相的体积由62％线性上升到100％。结果如图6所示；与出发菌株Coleophoma sp.-Δku80相比，化合物1、2、3消失，产生了少量的化合物6、7、8和9。通过LC-MS对Coleophoma sp.-ΔmcfS发酵产物进行分析。LC-MS分析方法为：Agilent公司1290高效液相色谱，色谱柱为Agilent Zorbax Extend-C18的C18柱(2.1×50mm，1.8μm)；流动相总流速为0.6mL/min；流动相A：0.05％(体积比)甲酸水溶液，流动相B：0.05％(体积比)甲酸乙腈溶液，总洗脱时间为7.0min；梯度洗脱条件：0-1min，流动相B占流动相的体积由5％线性上升到20％，1-6min，流动相B占流动相的体积由20％线性上升到60％，6-7min，流动相B占流动相的体积由60％线性上升到100％。结果如图7所示；通过LC-MS分析结果可知，当敲除基因mcfS之后，FR901379结构中的磺酰基消失，产生了化合物6、7、8和9。化合物6、7和8同样也在敲除株Coleophoma sp.-ΔmcfP中存在，这3个化合物有一个共同的特征，L-高酪氨酸苯环C3’位的氧磺酰基缺失。通过LC-MS对化合物9进行分析，化合物9分子式：C51H82N8O18，理论值：[M+H]+1095.5820实际值1095.5823，与化合物1相比，分子量小了80，推测应该少了磺酰基(SO3 -)；进一步通过NMR对其进行确认，发现化合物9在L-高酪氨酸苯环的C3’位只有羟基。以上结果表明McfS在FR901379生物合成中负责将磺酰基转移到L-高酪氨酸苯环的C3’位的羟基上。mcfS基因序列如SEQ ID No.3所示，McfS的氨基酸序列如SEQ ID No.4所示。
以McfS的氨基酸序列作为参照，在另外3株产其他磺酰化的棘白菌素类化合物生产菌株Coleophoma cylindrospora，Coleophoma crateriformis和Venustampullaechinocandica中找到了同源性大于75％的同源序列，在NCBI数据库中的序列号分别为RDW63433.1，RDW57264.1和XP_031866072.1。这3个氨基酸很有可能与McfS具有相同的功能，负责氧磺酰基模块中第二步磺酰基基团的形成。
实施例5产氧磺酰化纽莫康定B0的G.lozoyensis ATCC 74030工程菌株的构建
1.P450酶编码基因mcfP和磺酰基转移酶编码基因mcfS表达质粒构建
所述P450酶编码基因mcfP的基因序列如SEQ ID No.1所示，其氨基酸序列如SEQID No.2所示。所述磺酰基转移酶编码基因mcfS的基因序列如SEQ ID No.3所示，其氨基酸序列如SEQ ID No.4所示。
提取Coleophoma sp.MEFC009的RNA，对其RNA进行反转录获得cDNA。以反转录后的cDNA为模板，利用引物mcfPCDS-F(5’-cttattcctttgaacctttcaATGATAAATCTTGCAAGTCCCCTC-3’)和mcfPCDS-R(5’-caaaattcttcatttatttattatgcttccacaagtattcttaa-3’)进行PCR扩增，获得大小约为1.5kb的mcfP的编码序列(mcfPCDS)；以G.lozoyensis ATCC 74030基因组为模板，利用引物PgpdGL-F(5’-ctgggttcgcaaagataattgtgttactcatatggattgaggg-3’)和PgpdGL-R(5’-GGGGACTTGCAAGATTTATCATattgttttctggtgaagattag-3’)进行PCR扩增，获得大小约为1.0kb的启动子片段PgpdGL；以质粒pPM3为模板，利用引物Tpgk-F(5’-taaataaatgaagaattttgtgaaacgag-3’)和Tpgk-R(5’-cacacattattatggagaaacattgcagcgcacaagtcagt-3’)进行PCR扩增，获得大小约为0.5kb大小的终止子片段Tpgk；以质粒pXH2-1为模板，利用引物hph-F(5’-ttcgggatcgcaagcgtaaag-3’)和hph-R(5’-caattatctttgcgaacccagg-3’)进行PCR扩增，获得大小约为2.2kb的潮霉素抗性筛选片段hph；利用限制性内切酶Bam H I和Xho I对质粒pCAMBIA1300进行双酶切，纯化回收后获得线性的质粒pCAMBIA1300。利用一步克隆试剂盒(Ultra One Step CloningKit)将线性质粒pCAMBIA1300与片段hph，PgpdGL，mcfPCDS和Tpgk进行连接，获得重组质粒pCAMBIA1300-mcfP。将重组后的质粒pCAMBIA1300-mcfP转化到大肠杆菌DH5α感受态细胞，通过卡那霉素抗性筛选阳性转化子，通过PCR和DNA测序获得正确的重组质粒pCAMBIA1300-mcfP，质粒图谱如图8A。
参照上述相同的方法构建质粒pCAMBIA1300-mcfS。以反转录后的cDNA为模板，利用引物mcfSCDS-F(5’-caactcatcaatcatcacaacATGGCTTTAGACCGCCAGAATGC-3’)和mcfSCDS-R(5’-cacaaaattcttcatttatttaCTACTTCCTAGCTAGCCAAACAGCC-3’)进行PCR扩增，获得大小约为0.8kb的mcfS的编码序列(mcfSCDS)；以质粒pXH2-1为模板，利用引物PgpdAt-F(5’-ccctgggttcgcaaagataattggttacactctgggaggatcc-3’)和PgpdAt-R(5’-gttgtgatgattgatgagttg-3’)进行PCR扩增，获得大小约为0.7kb的启动子片段PgpdAt；利用一步克隆试剂盒(Ultra One Step Cloning Kit)将线性质粒pCAMBIA1300与片段hph、PgpdAt、mcfSCDS和Tpgk进行连接，获得重组质粒pCAMBIA1300-mcfS。将重组后的质粒pCAMBIA1300-mcfS转化到大肠杆菌DH5α感受态细胞，通过卡那霉素抗性筛选阳性转化子，通过PCR和DNA测序获得正确的重组质粒pCAMBIA1300-mcfS，质粒图谱如图8B。
2.产氧磺酰化纽莫康定B0菌株的构建
分别将重组质粒pCAMBIA1300-mcfP和pCAMBIA1300-mcfS转到农杆菌LBA4404感受态细胞，获得重组菌株LBA4404-pCAMBIA1300-mcfP和LBA4404-pCAMBIA1300-mcfS。通过根瘤农杆菌介导的转化，将片段hph-PgpdA-mcfP-Tpgk和hph-PgpdAt-mcfS-Tpgk共同转到两种方式转到G.lozoyensis ATCC 74030菌株中。
将具有潮霉素B抗性的转化子进行传代培养，连续传代3次，选取稳定传代的9个转化子进行分离纯化，25℃培养7-10天，对纯化后的单菌落提取基因组，分别利用引物PgpdGL-F(5’-ctgggttcgcaaagataattgtgttactcatatggattgaggg-3’)和mcfPCDS-R(5’-caaaattcttcatttatttattatgcttccacaagtattcttaa-3’)；PgpdAt-F(5’-ccctgggttcgcaaagataattggttacactctgggaggatcc-3’)和mcfSCDS-R(5’-cacaaaattcttcatttatttaCTACTTCCTAGCTAGCCAAACAGCC-3’)进行PCR验证，能同时扩增出大小约为3.0kb和2.1kb的条带为阳性转化子，由图9可知利用该方法获得的9个转化子为阳性转化子，基因组上同时整合了表达元件PgpdGL-mcfP-Tpgk和PgpdAt-mcfS-Tpgk，证明我们得到了表达mcfP和mcfS的G.lozoyensis菌株G.lozoyensis::mcfP::mcfS。
实施例6产氧磺酰化纽莫康定B0的G.lozoyensis::mcfP::mcfS工程菌株的发酵验证
将工程菌株G.lozoyensis::mcfP::mcfS和对照菌株G.lozoyensis ATCC 74030接种于PDA固体平板上，25℃培养7-10天。挑取少量的菌丝，利用核酸提取仪(-24)对菌丝进行破碎，将破碎后的菌丝接种于50mL G.lozoyensis ATCC 74030的种子培养基(250mL三角瓶)，25℃，220rpm，摇床培养4-5天。将上述培养的种子液取5mL到G.lozoyensisATCC 74030的发酵培养基，25℃，220rpm，摇床培养12天，每个菌株设置3个平行。从每一瓶发酵液中取1mL，加入等体积的甲醇，超声萃取1h，离心，取上清。用0.22μm的有机滤器过滤处理样品，并通过HPLC和LC-MS对处理后的样品进行分析。
HPLC分析方法为：液相色谱柱为Agilent C-18反向柱883975-902(4.6×150mm，5μm)；流动相为A：0.05％(体积比)三氟乙酸水溶液，流动相B：0.05％(体积比)三氟乙酸乙腈溶液，流速为1mL/min，紫外检测波长：210nm，30℃，总洗脱时间为25min。梯度洗脱条件：0-5min，流动相B占流动相的体积由5％线性上升到40％，5-20min，流动相B占流动相的体积由40％线性上升到62％，20-25min，流动相B占流动相的体积由62％线性上升到100％。结果如图10所示；从HPLC结果可知，除了化合物10(纽莫康定B0)外，在12.2min和13min出现了两个新的化合物，命名为化合物11和12。并且11和12和10具有相同的紫外吸收。猜测可能是羟化的纽莫康定B0和氧磺酰化纽莫康定B0的。为了进一步确认化合物的11和12结构，通过LC-MS进行分析。
LC-MS分析方法为：Agilent公司1290高效液相色谱，色谱柱为Agilent ZorbaxExtend-C18的C18柱(2.1×50mm，1.8μm)；流动相总流速为0.6mL/min；流动相A：0.05％(体积比)甲酸水溶液，流动相B：0.05％(体积比)甲酸乙腈溶液，总洗脱时间为7.5min；洗脱条件为：0-1min，流动相B占流动相的体积由5％线性上升到20％，1-6min，流动相B占流动相的体积由20％线性上升到60％，6-7min，流动相B占流动相的体积由60％线性上升到100％。结果如图11所示。从LC-MS分析结果可知，化合物11的质荷比[M+H]+为1081.5664(C50H80N8O18，理论值：1081.5663)，化合物12的质荷比[M+H]+为1161.5229(C50H80N8O21S，理论值：1161.5231)。进一步的NMR结果表明化合物11和12与化合物10相比(图12)，分别在L-高酪氨酸苯环的C3’位置多了一个羟基和一个氧磺酰基。说明P450酶McfP和McfS能够在G.lozoyensis ATCC 74030内催化纽莫康定B0生成氧磺酰化的纽莫康定B0。
实施例7产羟化纽莫康定B0的G.lozoyensis ATCC 74030工程菌株的构建
1.P450酶编码基因mcfP表达质粒构建
所述P450酶编码基因mcfP的基因序列如SEQ ID No.1所示，其氨基酸序列如SEQID No.2所示。提取Coleophoma sp.的RNA，对其RNA进行反转录获得cDNA。以反转录后的cDNA为模板，利用引物mcfPCDS-F(5’-cttattcctttgaacctttcaATGATAAATCTTGCAAGTCCCCTC-3’)和mcfPCDS-R(5’-caaaattcttcatttatttattatgcttccacaagtattcttaa-3’)进行PCR扩增，获得大小约为1.5kb的mcfP的编码序列(mcfPCDS)；以G.lozoyensis ATCC 74030基因组为模板，利用引物PgpdGL-F(5’-ctgggttcgcaaagataattgtgttactcatatggattgaggg-3’)和PgpdGL-R(5’-GGGGACTTGCAAGATTTATCATattgttttctggtgaagattag-3’)进行PCR扩增，获得大小约为1.0kb的启动子片段PgpdGL；以质粒pPM3基因组为模板，利用引物Tpgk-F(5’-taaataaatgaagaattttgtgaaacgag-3’)和Tpgk-R(5’-cacacattattatggagaaacattgcagcgcacaagtcagt-3’)进行PCR扩增，获得大小约为0.5kb大小的终止子片段Tpgk；以质粒pXH2-1为模板，利用引物hph-F(5’-ttcgggatcgcaagcgtaaag-3’)和hph-R(5’-caattatctttgcgaacccagg-3’)进行PCR扩增，获得大小约为2.2kb的潮霉素抗性筛选片段hph；利用限制性内切酶Bam HI和Xho I对质粒pCAMBIA1300进行双酶切，纯化回收后获得线性的质粒pCAMBIA1300。利用一步克隆试剂盒(Ultra One Step CloningKit)将线性质粒pCAMBIA1300与片段hph，PgpdGL，mcfPCDS和Tpgk进行连接，获得重组质粒pCAMBIA1300-mcfP。将重组后的质粒pCAMBIA1300-mcfP转化到大肠杆菌DH5α感受态细胞，通过卡那霉素抗性筛选阳性转化子，通过PCR和DNA测序获得正确的重组质粒pCAMBIA1300-mcfP，质粒图谱如图8A。
2.产羟化纽莫康定B0菌株的构建
分别将重组质粒pCAMBIA1300-mcfP转到农杆菌LBA4404感受态细胞，获得重组菌株LBA4404-pCAMBIA1300-mcfP通过根瘤农杆菌介导的转化，将片段hph-PgpdGL-mcfP-Tpgk转到G.lozoyensis ATCC 74030菌株中。
将具有潮霉素B抗性的转化子进行传代培养，连续传代3次，选取稳定传代的9个转化子进行分离纯化，25℃培养7-10天，对纯化后的单菌落提取基因组，分别利用引物PgpdGL-F(5’-ctgggttcgcaaagataattgtgttactcatatggattgaggg-3’)和mcfPCDS-R(5’-caaaattcttcatttatttattatgcttccacaagtattcttaa-3’)进行PCR验证，能同时扩增出大小约为2.5kb的条带为阳性转化子，由图13可知利用该方法获得15个阳性转化子，基因组上整合了表达元件PgpdGL-mcfP-Tpgk，证明我们得到了表达mcfP的G.lozoyensis菌株G.lozoyensis::mcfP。
实施例8产羟化纽莫康定B0的G.lozoyensis::mcfP工程菌株的发酵验证
将工程菌株G.lozoyensis::mcfP和对照菌株G.lozoyensis ATCC 74030接种于PDA固体平板上，25℃培养7-10天。挑取少量的菌丝，利用核酸提取仪(-24)对菌丝进行破碎，将破碎后的菌丝接种于50mL G.lozoyensis ATCC 74030的种子培养基(250mL三角瓶)，25℃，220rpm，摇床培养4-5天。将上述培养的种子液取5mL到G.lozoyensisATCC74030的发酵培养基，25℃，220rpm，摇床培养12天，每个菌株设置3个平行。从每一瓶发酵液中取1mL，加入等体积的甲醇，超声萃取1h，离心，取上清。用0.22μm的有机滤器过滤处理样品，并通过HPLC和LC-MS对处理后的样品进行分析。
HPLC分析方法为：液相色谱柱为Agilent C-18反向柱883975-902(4.6×150mm，5μm)；流动相为A：0.05％(体积比)三氟乙酸水溶液，流动相B：0.05％(体积比)三氟乙酸乙腈溶液，流速为1mL/min，紫外检测波长：210nm，30℃，总洗脱时间为25min。梯度洗脱条件：0-5min，流动相B占流动相的体积由5％线性上升到40％，5-20min，流动相B占流动相的体积由40％线性上升到62％，20-25min，流动相B占流动相的体积由62％线性上升到100％。结果如图14所示；从HPLC结果可知，除了化合物10(纽莫康定B0)外，在12.2min出现了一个新的化合物，命名为化合物11。并且11和10具有相同的紫外吸收。猜测可能是羟化的纽莫康定B0。为了进一步确认化合物的11结构，通过LC-MS进行分析。
LC-MS分析方法为：Agilent公司1290高效液相色谱，色谱柱为Agilent ZorbaxExtend-C18的C18柱(2.1×50mm，1.8μm)；流动相总流速为0.6mL/min；流动相A：0.05％(体积比)甲酸水溶液，流动相B：0.05％(体积比)甲酸乙腈溶液，总洗脱时间为7.5min；洗脱条件为：0-1min，流动相B占流动相的体积由5％线性上升到20％，1-6min，流动相B占流动相的体积由20％线性上升到60％，6-7min，流动相B占流动相的体积由60％线性上升到100％。结果如图11所示。从LC-MS分析结果可知，化合物11的质荷比[M+H]+为1081.5664(C50H80N8O18，理论值：1081.5663)。进一步的NMR结果表明化合物11与纽莫康定B0相比(图12)，在L-高酪氨酸苯环的C3’位置多了一个羟基。说明P450酶McfP能够在G.lozoyensis ATCC 74030内催化纽莫康定B0生成羟化的纽莫康定B0。
实施例9氧磺酰化纽莫康定B0水溶性分析
首先绘制纽莫康定B0、羟化纽莫康定B0(化合物11)和氧磺酰化的纽莫康定B0(化合物12)的标准曲线。将纽莫康定B0、化合物11和12称重后在25℃条件下分别加入少量的ddH2O，制备其饱和溶液，放置24h，使其充分溶解。将饱和溶液进行离心，12000rpm，15min，取上清通过HPLC进行分析。将饱和溶液的HPLC峰面积带入标准曲线，计算出化合物的溶解度，结果如图15所示；结果显示，化合物12(即氧磺酰化的纽莫康定B0)与纽莫康定B0和羟化纽莫康定B0相比，其水溶性能够显著的提升；并且氧磺酰化的纽莫康定B0比纽莫康定B0的水溶性提高了4000多倍。
HPLC分析方法：液相色谱柱为Agilent C-18反向柱883975-902(4.6×150mm，5μm)；流动相为A：0.05％(体积比)三氟乙酸水溶液，流动相B：0.05％(体积比)三氟乙酸乙腈溶液，流速为1mL/min，紫外检测波长：210nm，30℃，总洗脱时间为25min。梯度洗脱条件：0-5min，流动相B占流动相的体积由5％线性上升到40％，5-20min，流动相B占流动相的体积由40％线性上升到62％，20-25min，流动相B占流动相的体积由62％线性上升到100％。
实施例10氧磺酰化纽莫康定B0抗菌活性分析
将白色念珠菌(Canidia albicans[CMCC(F)98001])于5mL的PDB液体培养基中，25℃条件下培养1天至OD600＝1.0-3.0，将其稀释至OD600＝0.2-0.6，涂布到平板上。分别将含有两性霉素B、纽莫康定B0、羟化纽莫康定B0(化合物11)和氧磺酰化纽莫康定B0(化合物12)(终浓度为250μg/mL，DMSO溶解)的滤纸片放到长有白色念珠菌的PDA平板上，同时以加入相同体积的DMSO作为阴性对照，以两性霉素B作为阳性对照，25℃条件下培养1天。结果如图16所示，氧磺酰化纽莫康定B0与纽莫康定B0对白色念珠菌同样具有良好的抑制效果。
虽然本发明已以较佳的实施例公开如上，但其并非用以限定本发明，在不脱离本发明精神和范围内，本领域技术人员都可以在此基础上做出各种改动与变型，因此，本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 中国科学院青岛生物能源与过程研究所
<120> 氧磺酰化纽莫康定B0及其制备方法和应用
<130> 11
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1503
<212> DNA
<213> Artificial Sequence
<220>
<223> mcfP
<400> 1
atgataaatc ttgcaagtcc cctcttcgca acaacagcag ttctagtctg gctcagcagt 60
ctcataatct atcgcctata tctctctcca ctatctcgat ttcccggccc aaaactcgct 120
gctctaacag gatggtacga gacatacttc gacctcttta aacggggtcg ctactggatc 180
gagattgaac gcatgcacga agtctatggc cctatcatcc gcatcaatcc caatgagcta 240
catgttaatg acccagaatg gaatgagccc tacaagatca gcggccgcgt tgacaagtat 300
gactggtact acacctttgt tggtagttcc ggatcctcat ctgcattcgg aaccatagac 360
cacgacgttc atcgtggccg ccggaaagct caacagggct atttcaccac cgacgccatc 420
acgcgctttg aaccacattt agaaaccctg acagcaaagt tctgcgcaag actagacggc 480
ttcaagggga cgggaaagca tgttaatctc tccgatgcgt tccgatcaat cgcggtggat 540
gtggccgcga tgtttacatt gaatcaatcg tatggtttca tcgatgaccc ggatttcaag 600
gccgaggtcc atcaagggat ccgggcattt ccggatattg gagtgctgaa tcgccatttt 660
acgggtttgt tcgtggtttt ggagtcaatc catagatggg tgttgagtgt tatcaacccg 720
tcagaagaag ataatgggtt actcacaagt agaataaacc tgcattgtaa agctattatt 780
gccgactacg ccagtaagaa aggcgacgtc aagcccaata tcattcacag aatgctagac 840
gcaccagaac tatcgatgaa agataagaca gcgtggcgcc ttcaattgga ggcgcgcacc 900
cttataggag ctggaactga aacgacagga cacacattag ccgtcatagc attccatctg 960
ctagcaaatc cggagaaggc aaagaggttg aaggaggaga tcttagctac gaaagaaggg 1020
cgggaaaagc ctttaactta tcaggagtta caaatgcttc cgtatttatc ttctgtggtc 1080
cttgaaggtc atcgcatttc tagtgttgta tcaggtcgtc tgccacgggt caatacaaaa 1140
gagccgctca gatatggtga ctatagtatc cctattggca cacccgtcag caccacccaa 1200
cggttaacac actacaatgc caccatattc ccctccccaa acacattcct ccccgaacgt 1260
tggcttcagc cctcggaacg aaagcgcctg gagaaataca tccagccgtt cgggcgtggc 1320
tcaagatctt gtataggcat gcatcttgca aatgcagaga tttacaaaac attggcggag 1380
atgtttgcaa ggtttgacat gaagttatat gatacggagt tcgaggatat tatgcaagtg 1440
catgactttt ttacttcgtt tccatcgagc gagaggggtt taagaatact tgtggaagca 1500
taa 1503
<210> 2
<211> 500
<212> PRT
<213> Artificial Sequence
<220>
<223> mcfP
<400> 2
Met Ile Asn Leu Ala Ser Pro Leu Phe Ala Thr Thr Ala Val Leu Val
1 5 10 15
Trp Leu Ser Ser Leu Ile Ile Tyr Arg Leu Tyr Leu Ser Pro Leu Ser
20 25 30
Arg Phe Pro Gly Pro Lys Leu Ala Ala Leu Thr Gly Trp Tyr Glu Thr
35 40 45
Tyr Phe Asp Leu Phe Lys Arg Gly Arg Tyr Trp Ile Glu Ile Glu Arg
50 55 60
Met His Glu Val Tyr Gly Pro Ile Ile Arg Ile Asn Pro Asn Glu Leu
65 70 75 80
His Val Asn Asp Pro Glu Trp Asn Glu Pro Tyr Lys Ile Ser Gly Arg
85 90 95
Val Asp Lys Tyr Asp Trp Tyr Tyr Thr Phe Val Gly Ser Ser Gly Ser
100 105 110
Ser Ser Ala Phe Gly Thr Ile Asp His Asp Val His Arg Gly Arg Arg
115 120 125
Lys Ala Gln Gln Gly Tyr Phe Thr Thr Asp Ala Ile Thr Arg Phe Glu
130 135 140
Pro His Leu Glu Thr Leu Thr Ala Lys Phe Cys Ala Arg Leu Asp Gly
145 150 155 160
Phe Lys Gly Thr Gly Lys His Val Asn Leu Ser Asp Ala Phe Arg Ser
165 170 175
Ile Ala Val Asp Val Ala Ala Met Phe Thr Leu Asn Gln Ser Tyr Gly
180 185 190
Phe Ile Asp Asp Pro Asp Phe Lys Ala Glu Val His Gln Gly Ile Arg
195 200 205
Ala Phe Pro Asp Ile Gly Val Leu Asn Arg His Phe Thr Gly Leu Phe
210 215 220
Val Val Leu Glu Ser Ile His Arg Trp Val Leu Ser Val Ile Asn Pro
225 230 235 240
Ser Glu Glu Asp Asn Gly Leu Leu Thr Ser Arg Ile Asn Leu His Cys
245 250 255
Lys Ala Ile Ile Ala Asp Tyr Ala Ser Lys Lys Gly Asp Val Lys Pro
260 265 270
Asn Ile Ile His Arg Met Leu Asp Ala Pro Glu Leu Ser Met Lys Asp
275 280 285
Lys Thr Ala Trp Arg Leu Gln Leu Glu Ala Arg Thr Leu Ile Gly Ala
290 295 300
Gly Thr Glu Thr Thr Gly His Thr Leu Ala Val Ile Ala Phe His Leu
305 310 315 320
Leu Ala Asn Pro Glu Lys Ala Lys Arg Leu Lys Glu Glu Ile Leu Ala
325 330 335
Thr Lys Glu Gly Arg Glu Lys Pro Leu Thr Tyr Gln Glu Leu Gln Met
340 345 350
Leu Pro Tyr Leu Ser Ser Val Val Leu Glu Gly His Arg Ile Ser Ser
355 360 365
Val Val Ser Gly Arg Leu Pro Arg Val Asn Thr Lys Glu Pro Leu Arg
370 375 380
Tyr Gly Asp Tyr Ser Ile Pro Ile Gly Thr Pro Val Ser Thr Thr Gln
385 390 395 400
Arg Leu Thr His Tyr Asn Ala Thr Ile Phe Pro Ser Pro Asn Thr Phe
405 410 415
Leu Pro Glu Arg Trp Leu Gln Pro Ser Glu Arg Lys Arg Leu Glu Lys
420 425 430
Tyr Ile Gln Pro Phe Gly Arg Gly Ser Arg Ser Cys Ile Gly Met His
435 440 445
Leu Ala Asn Ala Glu Ile Tyr Lys Thr Leu Ala Glu Met Phe Ala Arg
450 455 460
Phe Asp Met Lys Leu Tyr Asp Thr Glu Phe Glu Asp Ile Met Gln Val
465 470 475 480
His Asp Phe Phe Thr Ser Phe Pro Ser Ser Glu Arg Gly Leu Arg Ile
485 490 495
Leu Val Glu Ala
500
<210> 3
<211> 849
<212> DNA
<213> Artificial Sequence
<220>
<223> mcfS
<400> 3
atggctttag accgccagaa tgcgaaagtt acaactttcg gtctgtcaaa gccgaaaacc 60
aatatagatc gccgatcatg tcagagaact gtccccatga aggttctctg cctaggacta 120
tgtcgaaccg gcacttcctc attgcgtgcg gctctctttg agcttggcct tgatgatgtc 180
tatcacatgt gtagtgtgac ggaagagaat cccctcgact ccaagttgtg gaaagaggcc 240
ttcgacgcga aatatgaagg gatcggcaag ccctacggaa gagctgaatt tgacgcactc 300
ttgggtcatt gcatggcaac ctcggatttc cccagcgttg ccttcgctcc agaactcatc 360
gccgcttacc ccgaggcaaa gataattctc actgtacgag ataacgccga tgtctggtat 420
gactccgttc tcaacacgat ctggagagtc tccaacttcc ttcgcgctcc tccgagaact 480
ttaacccaac gagtcgttca agcgattctt cccaagccgg atttcaacat attcaagtac 540
agcccccttg gcaactttcc tgaggaaggc tgtcagtggt atagtgactg gaatgaagag 600
attagaactc tagccaaagg gagggacttc ttggaattca atgtaaagga gggatggggt 660
ccactctgta gattcttgga ggtggagcag ccggagacgc catttccaag agtcaatgat 720
tcaaatacat tcaaggaatt tcatgataag ggtttggagc aggatattca aagactggta 780
ggcataagta ctaagcttgt cgccgctgtt ggtgtattgg gtttggctgt ttggctagct 840
aggaagtag 849
<210> 4
<211> 282
<212> PRT
<213> Artificial Sequence
<220>
<223> mcfS
<400> 4
Met Ala Leu Asp Arg Gln Asn Ala Lys Val Thr Thr Phe Gly Leu Ser
1 5 10 15
Lys Pro Lys Thr Asn Ile Asp Arg Arg Ser Cys Gln Arg Thr Val Pro
20 25 30
Met Lys Val Leu Cys Leu Gly Leu Cys Arg Thr Gly Thr Ser Ser Leu
35 40 45
Arg Ala Ala Leu Phe Glu Leu Gly Leu Asp Asp Val Tyr His Met Cys
50 55 60
Ser Val Thr Glu Glu Asn Pro Leu Asp Ser Lys Leu Trp Lys Glu Ala
65 70 75 80
Phe Asp Ala Lys Tyr Glu Gly Ile Gly Lys Pro Tyr Gly Arg Ala Glu
85 90 95
Phe Asp Ala Leu Leu Gly His Cys Met Ala Thr Ser Asp Phe Pro Ser
100 105 110
Val Ala Phe Ala Pro Glu Leu Ile Ala Ala Tyr Pro Glu Ala Lys Ile
115 120 125
Ile Leu Thr Val Arg Asp Asn Ala Asp Val Trp Tyr Asp Ser Val Leu
130 135 140
Asn Thr Ile Trp Arg Val Ser Asn Phe Leu Arg Ala Pro Pro Arg Thr
145 150 155 160
Leu Thr Gln Arg Val Val Gln Ala Ile Leu Pro Lys Pro Asp Phe Asn
165 170 175
Ile Phe Lys Tyr Ser Pro Leu Gly Asn Phe Pro Glu Glu Gly Cys Gln
180 185 190
Trp Tyr Ser Asp Trp Asn Glu Glu Ile Arg Thr Leu Ala Lys Gly Arg
195 200 205
Asp Phe Leu Glu Phe Asn Val Lys Glu Gly Trp Gly Pro Leu Cys Arg
210 215 220
Phe Leu Glu Val Glu Gln Pro Glu Thr Pro Phe Pro Arg Val Asn Asp
225 230 235 240
Ser Asn Thr Phe Lys Glu Phe His Asp Lys Gly Leu Glu Gln Asp Ile
245 250 255
Gln Arg Leu Val Gly Ile Ser Thr Lys Leu Val Ala Ala Val Gly Val
260 265 270
Leu Gly Leu Ala Val Trp Leu Ala Arg Lys
275 280
Claims (10)
1.一种氧磺酰化的纽莫康定B0，所述氧磺酰化纽莫康定B0的结构式如式(I)所示：
2.一种纽莫康定产生菌(Glarea lozoyensis)的基因工程菌株，所述工程菌株为在纽莫康定产生菌(G.lozoyensis)中引入细胞色素P450单加氧酶和磺酰基转移酶所得到的工程菌株；
所述细胞色素P450单加氧酶与SEQ ID No.2相比具有至少70％的序列同一性；
所述磺酰基转移酶与SEQ ID No.4相比具有至少70％的序列同一性。
3.一种制备权利要求1所述的氧磺酰化的纽莫康定B0的方法，所述方法包括利用权利要求2所述的基因工程菌株进行发酵的步骤。
4.权利要求2中所述的细胞色素P450单加氧酶和磺酰基转移酶，或其编码基因在制备生产权利要求1所述的氧磺酰化的纽莫康定B0的基因工程菌株中的应用，所述基因工程菌株的出发菌株为纽莫康定产生菌(G.lozoyensis)。
5.权利要求2中所述的细胞色素P450单加氧酶和磺酰基转移酶，或者包含权利要求2中所述的细胞色素P450单加氧酶和磺酰基转移酶的生物材料，或者权利要求2所述的基因工程菌株在制备权利要求1所述的氧磺酰化的纽莫康定B0中的应用。
6.根据权利要求5所述的应用，其特征在于，所述生物材料选自：包含权利要求2中所述的细胞色素P450单加氧酶和磺酰基转移酶，或其编码基因的载体；或者，包含权利要求2中所述的细胞色素P450单加氧酶和磺酰基转移酶，或其编码基因的宿主细胞。
7.权利要求2中所述的细胞色素P450单加氧酶和磺酰基转移酶在催化纽莫康定B0形成权利要求1所述的氧磺酰化的纽莫康定B0中的应用。
8.一种制备权利要求1所述的氧磺酰化的纽莫康定B0的方法，所述方法包括利用权利要求2中所述的细胞色素P450单加氧酶和磺酰基转移酶催化纽莫康定B0形成所述氧磺酰化的纽莫康定B0中的步骤。
9.权利要求1所述的氧磺酰化的纽莫康定B0，或者权利要求3或8所述方法制备得到的氧磺酰化的纽莫康定B0在制备抗真菌药物中的用途。
10.一种抗真菌药物，所述药物包括权利要求1所述的氧磺酰化的纽莫康定B0，或者权利要求3或8所述方法制备得到的氧磺酰化的纽莫康定B0。
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