CN117105886A - Tetracyclic sesquiterpenoids and their synthetic gene clusters - Google Patents
Tetracyclic sesquiterpenoids and their synthetic gene clusters Download PDFInfo
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- CN117105886A CN117105886A CN202310985911.3A CN202310985911A CN117105886A CN 117105886 A CN117105886 A CN 117105886A CN 202310985911 A CN202310985911 A CN 202310985911A CN 117105886 A CN117105886 A CN 117105886A
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
- C07C49/20—Unsaturated compounds containing keto groups bound to acyclic carbon atoms
- C07C49/24—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing hydroxy groups
- C07C49/242—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing hydroxy groups containing rings other than six-membered aromatic rings
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/02—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen
- C07C69/12—Acetic acid esters
- C07C69/21—Acetic acid esters of hydroxy compounds with more than three hydroxy groups
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- C07C69/614—Esters of carboxylic acids having a carboxyl group bound to an acyclic carbon atom and having a six-membered aromatic ring in the acid moiety of phenylacetic acid
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- C07D303/14—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by free hydroxyl radicals
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- C07D303/32—Compounds containing oxirane rings with hydrocarbon radicals, substituted by singly or doubly bound oxygen atoms by aldehydo- or ketonic radicals
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- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/04—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
- C07D307/18—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/20—Oxygen atoms
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Abstract
Description
技术领域Technical field
本发明涉及基因工程领域，尤其是涉及四环二倍半萜类化合物及其合成基因簇。The present invention relates to the field of genetic engineering, in particular to tetracyclic sesquiterpenoid compounds and their synthetic gene clusters.
背景技术Background technique
真菌天然产物是药物开发的重要来源，大量基因组的解密揭示真菌含有大量潜在的新颖生物合成基因簇“暗物质”。萜类化合物是所有异戊二烯聚合物及其衍生物的总称，丰富的结构类型及广泛的生物活性决定了其在天然药物研究中的重要地位。其中二萜和二倍半萜是真菌中结构类型最丰富多样和最重要的次级代谢产物，广泛应用于制药、香料、树脂和其他精细化学品。随着NCBI中释放基因组数据库的不断增加，研究者发现真菌基因组中蕴含着大量萜类生物合成基因簇，这就意味着以萜类合酶基因组挖掘为导向，通过代谢工程和合成生物学策略，在微生物体内重构代谢途径，不仅能够获得更多新结构、高活性的化合物，还能发现不同种类化合物的生物合成途径，这为新药研发提供崭新的途径。Fungal natural products are an important source of drug development, and the decryption of large genomes has revealed that fungi contain a large number of potentially novel biosynthetic gene clusters "dark matter." Terpenoids are the general term for all isoprene polymers and their derivatives. Their rich structural types and wide range of biological activities determine their important position in natural medicine research. Among them, diterpenes and sesquiterpenes are the most diverse and important secondary metabolites in fungi and are widely used in pharmaceuticals, spices, resins and other fine chemicals. With the increasing number of released genome databases in NCBI, researchers have found that fungal genomes contain a large number of terpene biosynthetic gene clusters, which means that guided by terpene synthase genome mining, through metabolic engineering and synthetic biology strategies, Reconstructing metabolic pathways in microorganisms can not only obtain more compounds with new structures and high activity, but also discover the biosynthetic pathways of different types of compounds, which provides a new way for the development of new drugs.
Mangicols是一类具有多种潜在药用价值的四环二倍半萜类化合物，如mangicolsA和B表现出显著的抗炎活性，并对多种癌细胞系具有良好的细胞毒活性。自2000年首次报道，迄今为止仅发现了13个Mangicols类化合物。因此Mangicols类化合物具有较好的开发及研究前景。通过对其生物合成基因簇进行异源表达，可有效避免传统天然产物研究中的盲目性，高效挖掘更多具有生物活性的Mangicols类化合物。Mangicols are a class of tetracyclic bisquiterpenoids with multiple potential medicinal values, such as mangicols A and B, which exhibit significant anti-inflammatory activity and good cytotoxic activity against a variety of cancer cell lines. Since first reported in 2000, only 13 mangicols have been discovered so far. Therefore, Mangicols compounds have good development and research prospects. By heterologous expression of their biosynthetic gene clusters, blindness in traditional natural product research can be effectively avoided, and more biologically active Mangicols compounds can be efficiently mined.
发明内容Contents of the invention
针对现有技术的不足，本发明要解决的问题是提供四环二倍半萜类化合物及其合成基因簇。In view of the shortcomings of the existing technology, the problem to be solved by the present invention is to provide tetracyclic bisquiterpenoid compounds and their synthetic gene clusters.
本发明的目的可以通过以下技术方案来实现：The object of the present invention can be achieved through the following technical solutions:
本发明首先提供一类四环二倍半萜类化合物，Mangicol H至Mangicol P的结构式分别如下所示：The present invention first provides a class of tetracyclic bisquiterpenoids. The structural formulas of Mangicol H to Mangicol P are as follows:
本发明进一步提供四环二倍半萜类化合物Mangicol H至Mangicol P的生物合成基因簇fomd，含有6个基因，分别为编码二倍半萜合酶FoMS的基因fomdE或其功能等同体，编码细胞色素P450酶fomdA、fomdC、fomdD的基因fomdA，fomdC，fomdD或其功能等同体，编码醛酮还原酶fomdB的基因fomdB或其功能等同体，编码水解酶fomdF的基因fomdF或其功能等同体，其中，所述fomdA的核苷酸序列如SEQ ID NO.1所示，fomdB的核苷酸序列如SEQ ID NO.2所示，fomdC的核苷酸序列如SEQ ID NO.3所示，fomdD的核苷酸序列如SEQ ID NO.4所示，fomdF的核苷酸序列如SEQ ID NO.5所示，fomdE的核苷酸序列如SEQ ID NO.6所示；The present invention further provides a biosynthetic gene cluster fomd of the tetracyclic sesquiterpenoids Mangicol H to Mangicol P, which contains six genes, respectively the gene fomdE encoding the sesquiterpene synthase FoMS or its functional equivalent, encoding cells The genes fomdA, fomdC, fomdD or their functional equivalents of the pigment P450 enzymes fomdA, fomdC, fomdD, the gene fomdB encoding the aldehyde-keto reductase fomdB or their functional equivalents, the gene fomdF encoding the hydrolase fomdF or its functional equivalents, wherein , the nucleotide sequence of fomdA is shown in SEQ ID NO.1, the nucleotide sequence of fomdB is shown in SEQ ID NO.2, the nucleotide sequence of fomdC is shown in SEQ ID NO.3, and the nucleotide sequence of fomdD The nucleotide sequence is shown in SEQ ID NO.4, the nucleotide sequence of fomdF is shown in SEQ ID NO.5, and the nucleotide sequence of fomdE is shown in SEQ ID NO.6;
或者，6个基因的核苷酸序列是分别与编码蛋白fomdA、fomdB、fomdC、fomdD、fomdF和FoMS的氨基酸序列一致性高于80％所对应的DNA编码序列。Alternatively, the nucleotide sequences of the six genes are DNA coding sequences corresponding to an amino acid sequence identity of more than 80% with the coding proteins fomdA, fomdB, fomdC, fomdD, fomdF and FoMS respectively.
本发明进一步提供四环二倍半萜类化合物Mangicol H至Mangicol P的生物合成基因簇fomd在制备萜类化合物中的应用。The present invention further provides the application of the biosynthetic gene cluster fomd of the tetracyclic bisquiterpenoid compounds Mangicol H to Mangicol P in the preparation of terpenoid compounds.
在本发明的一个实施方式中，提供四环二倍半萜类化合物Mangicol H至MangicolP的生物合成基因簇fomd在合成四环二倍半萜类化合物Mangicol H至Mangicol P中的应用。In one embodiment of the present invention, the application of the biosynthetic gene cluster fomd of the tetracyclic sesquiterpenoids Mangicol H to Mangicol P in the synthesis of the tetracyclic sesquiterpenoids Mangicol H to Mangicol P is provided.
本发明进一步提供表达四环二倍半萜类化合物Mangicol H至Mangicol P的生物合成基因簇fomd的载体，为含有基因fomdA、fomdB、fomdC、fomdD、fomdF和fomdE的真核或原核表达载体。The present invention further provides a vector expressing the biosynthetic gene cluster fomd of the tetracyclic sesquiterpenoids Mangicol H to Mangicol P, which is a eukaryotic or prokaryotic expression vector containing the genes fomdA, fomdB, fomdC, fomdD, fomdF and fomdE.
本发明进一步提供所述四环二倍半萜类化合物的制备方法，通过米曲霉Aspergillus oryzae NSAR1中异源表达的方法表达尖孢镰刀菌14005(Fusariumoxysporum 14005)中四环二倍半萜类化合物Mangicol H至Mangicol P的生物合成基因簇fomd中的基因来获得四环二倍半萜类化合物Mangicol H至Mangicol P。The present invention further provides a preparation method of the tetracyclic sesquiterpenoid compound, which expresses the tetracyclic sesquiterpenoid compound Mangicol in Fusarium oxysporum 14005 through the heterologous expression method in Aspergillus oryzae NSAR1. The genes in the biosynthetic gene cluster fomd from H to Mangicol P are used to obtain the tetracyclic bisquiterpenoids Mangicol H to Mangicol P.
其中，米曲霉Aspergillus oryzae NSAR1、尖孢镰刀菌14005(Fusariumoxysporum14005)均为本领域技术人员已知的生物材料。Among them, Aspergillus oryzae NSAR1 and Fusarium oxysporum 14005 are biological materials known to those skilled in the art.
在本发明的一个实施方式中，所述四环二倍半萜类化合物的制备方法包括以下步骤：In one embodiment of the present invention, the preparation method of the tetracyclic sesquiterpenoid compound includes the following steps:
(1)以Fusarium oxysporum 14005的基因组为模板，分别以引物fomdE-F/fomdE-R，fomdA-F/fomdA-R，fomdC-F/fomdC-R，fomdD-F/fomdD-R，fomdB-F/fomdB-R和fomdF-F/fomdF-R对二倍半萜合酶的基因fomdE，细胞色素P450酶基因fomdA，fomdC，fomdD，醛酮还原酶基因fomdB，水解酶基因fomdF，进行PCR扩增，得到基因fomdE，fomdA，fomdC，fomdD，fomdB和fomdF的PCR产物；然后以米曲霉Aspergillus oryzae NSAR1表达载体pUARA4为载体构建fomdE，fomdA和fomdC的共表达载体pUARA4-fomdACE；以米曲霉Aspergillus oryzae NSAR1表达载体pUSA4为载体构建fomdD，fomdB和fomdF的共表达载体pUSA4-fomdBDF；(1) Using the genome of Fusarium oxysporum 14005 as a template, use primers fomdE-F/fomdE-R, fomdA-F/fomdA-R, fomdC-F/fomdC-R, fomdD-F/fomdD-R, fomdB-F respectively. /fomdB-R and fomdF-F/fomdF-R perform PCR amplification of the bisquiterpene synthase gene fomdE, cytochrome P450 enzyme genes fomdA, fomdC, fomdD, aldehyde-keto reductase gene fomdB, and hydrolase gene fomdF. , obtain the PCR products of genes fomdE, fomdA, fomdC, fomdD, fomdB and fomdF; then use Aspergillus oryzae NSAR1 expression vector pUARA4 as the vector to construct fomdE, fomdA and fomdC co-expression vector pUARA4-fomdACE; use Aspergillus oryzae NSAR1 The expression vector pUSA4 is used to construct the co-expression vector pUSA4-fomdBDF of fomdD, fomdB and fomdF;
(2)在PEG溶剂的介导下，将共表达载体pUARA4-fomdACE和pUSA4-fomdBDF共同转化至易于表达萜类合酶基因的高产宿主米曲霉A.oryzae NSAR1(niaD-，sC-，ΔargB，adeA-)的原生质体中，获得能产生四环二倍半萜类化合物Mangicol H至Mangicol P的米曲霉转化子AO-fomdABCDEF；(2) Under the mediation of PEG solvent, the co-expression vectors pUARA4-fomdACE and pUSA4-fomdBDF were co-transformed into the high-yield host Aspergillus oryzae NSAR1 (niaD-, sC-, ΔargB, which is easy to express terpene synthase genes). From the protoplasts of adeA-), the Aspergillus oryzae transformant AO-fomdABCDEF that can produce the tetracyclic bisquiterpenoid compounds Mangicol H to Mangicol P was obtained;
(3)将米曲霉转化子AO-fomdABCDEF的菌丝体接种并培养，生成四环二倍半萜类化合物Mangicol H至Mangicol P。(3) Inoculate and culture the mycelium of Aspergillus oryzae transformant AO-fomdABCDEF to generate tetracyclic bisquiterpenoid compounds Mangicol H to Mangicol P.
在本发明的一个实施方式中，所述引物fomdA-F/fomdA-R的核苷酸序列分别如SEQID NO.7、8所示，所述fomdB-F/fomdB-R的核苷酸序列分别如SEQ ID NO.9、10所示，所述fomdC-F/fomdC-R的核苷酸序列分别如SEQ ID NO.11、12所示，所述fomdD-F/fomdD-R的核苷酸序列分别如SEQ ID NO.13、14所示，所述fomdE-F/fomdE-R的核苷酸序列分别如SEQ IDNO.15、16所示，所述fomdF-F/fomdF-R的核苷酸序列分别是如SEQ ID NO.17、18所示。In one embodiment of the present invention, the nucleotide sequences of the primers fomdA-F/fomdA-R are shown in SEQ ID NO. 7 and 8 respectively, and the nucleotide sequences of the fomdB-F/fomdB-R are respectively As shown in SEQ ID NO.9 and 10, the nucleotide sequence of fomdC-F/fomdC-R is shown in SEQ ID NO.11 and 12 respectively, and the nucleotide sequence of fomdD-F/fomdD-R The sequences are shown in SEQ ID NO.13 and 14 respectively. The nucleotide sequences of fomdE-F/fomdE-R are shown in SEQ ID NO.15 and 16 respectively. The nucleosides of fomdF-F/fomdF-R are shown in SEQ ID NO.15 and 16 respectively. The acid sequences are shown in SEQ ID NO. 17 and 18 respectively.
在本发明的一个实施方式中，步骤(3)中，将米曲霉转化子AO-fomdABCDEF的菌丝体接种并培养的方法为：将米曲霉转化子AO-fomdABCDEF的菌丝体接种于含0.1％腺嘌呤的MPY培养基中，于30℃，220rpm培养2天作为种子液，按照80g大米，120mL去离子水中加入5mL种子液的比例，接种于添加0.1％腺嘌呤的大米固体培养基中，30℃，静置培养18天。In one embodiment of the present invention, in step (3), the method of inoculating and cultivating the mycelium of the Aspergillus oryzae transformant AO-fomdABCDEF is: inoculating the mycelium of the Aspergillus oryzae transformant AO-fomdABCDEF in a solution containing 0.1 % adenine in MPY medium, cultured at 30°C, 220rpm for 2 days as seed liquid, according to the ratio of 80g rice, 120mL deionized water adding 5mL seed liquid, inoculated into rice solid medium supplemented with 0.1% adenine, Incubate at 30°C for 18 days.
在本发明的一个实施方式中，步骤(3)中，将米曲霉转化子AO-fomdABCDEF的菌丝体接种并培养后，将AO-fomdABCDEF的固体发酵物加入等体积的乙酸乙酯萃取3次，蒸干后获得浸膏；浸膏用石油醚萃取获得极性小的组分，石油醚萃取液蒸干后进行正相分离，以石油醚和乙酸乙酯为流动相进行洗脱并富集目标组分Fr.5和Fr.7；Fr.5进行凝胶柱分离，最终用反相Cholester色谱柱，乙腈/0.1％甲酸水体积比80:20为流动相洗脱获得MangicolK；Fr.7进行凝胶柱分离获得三个馏分Fr.7.1-Fr.7.3.Fr.7.2用ACE C18-PFP色谱柱进行半制备，以乙腈/0.1％甲酸水体积比75:25为流动相洗脱获得Mangicol O和馏分Fr.7.2.1；馏分Fr.7.2.1进一步用手型柱Chiralpak IA，以正己烷/乙醇体积比95：5为流动相获得Mangicols H和I，Fr.7.3用Phenomenex色谱柱进行半制备，以乙腈/0.1％甲酸水体积比80:20为流动相洗脱获得Mangicol L，MangicolN和Mangicol P以及馏分Fr.7.3.1；馏分Fr.7.3.1进一步用手型柱Chiralpak IA，以正己烷/乙醇体积比94：6为流动相获得Mangicol J和Mangicol M。In one embodiment of the present invention, in step (3), after the mycelium of the Aspergillus oryzae transformant AO-fomdABCDEF is inoculated and cultured, the solid fermentation product of AO-fomdABCDEF is added to an equal volume of ethyl acetate and extracted three times. , obtain the extract after evaporating to dryness; the extract is extracted with petroleum ether to obtain small polar components. The petroleum ether extract is evaporated to dryness and then subjected to normal phase separation. Petroleum ether and ethyl acetate are used as mobile phases for elution and enrichment. The target components Fr.5 and Fr.7; Fr.5 were separated on a gel column, and finally a reversed-phase Cholester chromatographic column was used to elute with an acetonitrile/0.1% formic acid water volume ratio of 80:20 as the mobile phase to obtain MangicolK; Fr.7. Perform gel column separation to obtain three fractions Fr.7.1-Fr.7.3.Fr.7.2. Use ACE C18-PFP chromatographic column for semi-preparation. Use acetonitrile/0.1% formic acid water volume ratio 75:25 as the mobile phase to obtain Mangicol. O and fraction Fr.7.2.1; fraction Fr.7.2.1 was further processed with a hand-type column Chiralpak IA, using n-hexane/ethanol volume ratio 95:5 as the mobile phase to obtain Mangicols H and I, and Fr.7.3 was carried out with a Phenomenex chromatographic column. Semi-preparative, using acetonitrile/0.1% formic acid to water volume ratio of 80:20 as the mobile phase to obtain Mangicol L, MangicolN and Mangicol P as well as fraction Fr.7.3.1; fraction Fr.7.3.1 was further used to hand-type column Chiralpak IA, Mangicol J and Mangicol M were obtained using n-hexane/ethanol volume ratio 94:6 as the mobile phase.
与现有技术相比，本发明具有以下优点及有益效果：Compared with the existing technology, the present invention has the following advantages and beneficial effects:
本发明提供了一类四环二倍半萜类化合物及其制备方法与应用。研发过程中发明人通过基因组挖掘的策略从尖孢镰刀菌14005(Fusarium oxysporum 14005)中发现了新颖的Mangicols H-P生物合成基因簇。通过异源表达的方式，实现了Mangicols H-P生物合成基因簇中的基因在米曲霉中的表达。最终，分离纯化获得了9个全新的四环二倍半萜类化合物，即四环二倍半萜类化合物Mangicol H至Mangicol P。The invention provides a class of tetracyclic sesquiterpenoid compounds and their preparation methods and applications. During the research and development process, the inventors discovered a novel Mangicols H-P biosynthetic gene cluster from Fusarium oxysporum 14005 through a genome mining strategy. Through heterologous expression, the genes in the Mangicols H-P biosynthetic gene cluster were expressed in Aspergillus oryzae. Finally, nine new tetracyclic sesquiterpenoids were separated and purified, namely tetracyclic sesquiterpenoids Mangicol H to Mangicol P.
本发明的创新点主要体现在利用基因工程的方法，构建产生新四环二倍半萜类化合物Mangicol H至Mangicol P的自给自足工程菌。整个操作过程简单，工艺成熟，成本低廉，整个过程不含有害杂质，无毒，对环境非常友好。获得的产物Mangicols H-P为二倍半萜化合物的生物合成提供了新的资源，为该类化合物种类的获得提供了一种选择，为丰富天然产物化合物库、发现新抗生素提供了宝贵的先导化合物资源。The innovation point of the present invention is mainly reflected in the use of genetic engineering methods to construct self-sufficient engineering bacteria that produce new tetracyclic bisquiterpenoids Mangicol H to Mangicol P. The entire operation process is simple, the technology is mature, and the cost is low. The entire process does not contain harmful impurities, is non-toxic, and is very environmentally friendly. The obtained product Mangicols H-P provides a new resource for the biosynthesis of squiterpene compounds, provides a choice for the acquisition of this type of compounds, and provides valuable lead compound resources for enriching the natural product compound library and discovering new antibiotics. .
附图说明Description of drawings
图1为本发明化合物Mangicols H-P的紫外吸收谱图。Figure 1 is the ultraviolet absorption spectrum of the compound Mangicols H-P of the present invention.
图2为本发明化合物Mangicol H的HR-EI-MS谱图。Figure 2 is the HR-EI-MS spectrum of the compound Mangicol H of the present invention.
图3为本发明化合物Mangicol I的HR-EI-MS谱图。Figure 3 is the HR-EI-MS spectrum of the compound Mangicol I of the present invention.
图4为本发明化合物Mangicol J的HR-EI-MS谱图。Figure 4 is the HR-EI-MS spectrum of the compound Mangicol J of the present invention.
图5为本发明化合物Mangicol K的HR-EI-MS谱图。Figure 5 is the HR-EI-MS spectrum of the compound Mangicol K of the present invention.
图6为本发明化合物Mangicol L的HR-EI-MS谱图。Figure 6 is the HR-EI-MS spectrum of the compound Mangicol L of the present invention.
图7为本发明化合物Mangicol M的HR-EI-MS谱图。Figure 7 is the HR-EI-MS spectrum of the compound Mangicol M of the present invention.
图8为本发明化合物Mangicol N的HR-EI-MS谱图。Figure 8 is the HR-EI-MS spectrum of the compound Mangicol N of the present invention.
图9为本发明化合物Mangicol O的HR-EI-MS谱图。Figure 9 is the HR-EI-MS spectrum of the compound Mangicol O of the present invention.
图10为本发明化合物Mangicol P的HR-EI-MS谱图。Figure 10 is the HR-EI-MS spectrum of the compound Mangicol P of the present invention.
图11为本发明化合物Mangicol H溶于pyridine-d5中的1H-NMR谱图。Figure 11 is a 1 H-NMR spectrum of the compound Mangicol H of the present invention dissolved in pyridine-d 5 .
图12为本发明化合物Mangicol I溶于pyridine-d5中的1H-NMR谱图。Figure 12 is a 1 H-NMR spectrum of the compound Mangicol I of the present invention dissolved in pyridine-d 5 .
图13为本发明化合物Mangicol J溶于pyridine-d5中的1H-NMR谱图。Figure 13 is a 1 H-NMR spectrum of the compound Mangicol J of the present invention dissolved in pyridine-d 5 .
图14为本发明化合物Mangicol K溶于pyridine-d5中的1H-NMR谱图。Figure 14 is a 1 H-NMR spectrum of the compound Mangicol K of the present invention dissolved in pyridine-d 5 .
图15为本发明化合物Mangicol L溶于pyridine-d5中的1H-NMR谱图。Figure 15 is a 1 H-NMR spectrum of the compound Mangicol L of the present invention dissolved in pyridine-d 5 .
图16为本发明化合物Mangicol M溶于pyridine-d5中的1H-NMR谱图。Figure 16 is a 1 H-NMR spectrum of the compound Mangicol M of the present invention dissolved in pyridine-d 5 .
图17为本发明化合物Mangicol N溶于pyridine-d5中的1H-NMR谱图。Figure 17 is a 1 H-NMR spectrum of the compound Mangicol N of the present invention dissolved in pyridine-d 5 .
图18为本发明化合物Mangicol O溶于pyridine-d5中的1H-NMR谱图。Figure 18 is a 1 H-NMR spectrum of the compound Mangicol O of the present invention dissolved in pyridine-d 5 .
图19为本发明化合物Mangicol P溶于pyridine-d5中的1H-NMR谱图。Figure 19 is a 1 H-NMR spectrum of the compound Mangicol P of the present invention dissolved in pyridine-d 5 .
图20为本发明化合物Mangicol H溶于pyridine-d5中的13C-NMR谱图。Figure 20 is a 13 C-NMR spectrum of the compound Mangicol H of the present invention dissolved in pyridine-d 5 .
图21为本发明化合物Mangicol I溶于pyridine-d5中的13C-NMR谱图。Figure 21 is a 13 C-NMR spectrum of the compound Mangicol I of the present invention dissolved in pyridine-d 5 .
图22为本发明化合物Mangicol J溶于pyridine-d5中的13C-NMR谱图。Figure 22 is a 13 C-NMR spectrum of the compound Mangicol J of the present invention dissolved in pyridine-d 5 .
图23为本发明化合物Mangicol K溶于pyridine-d5中的13C-NMR谱图。Figure 23 is a 13 C-NMR spectrum of the compound Mangicol K of the present invention dissolved in pyridine-d 5 .
图24为本发明化合物Mangicol L溶于pyridine-d5中的13C-NMR谱图。Figure 24 is a 13 C-NMR spectrum of the compound Mangicol L of the present invention dissolved in pyridine-d 5 .
图25为本发明化合物Mangicol M溶于pyridine-d5中的13C-NMR谱图。Figure 25 is a 13 C-NMR spectrum of the compound Mangicol M of the present invention dissolved in pyridine-d 5 .
图26为本发明化合物Mangicol N溶于pyridine-d5中的13C-NMR谱图。Figure 26 is a 13 C-NMR spectrum of the compound Mangicol N of the present invention dissolved in pyridine-d 5 .
图27为本发明化合物Mangicol O溶于pyridine-d5中的13C-NMR谱图。Figure 27 is a 13 C-NMR spectrum of the compound Mangicol O of the present invention dissolved in pyridine-d 5 .
图28为本发明化合物Mangicol P溶于pyridine-d5中的13C-NMR谱图。Figure 28 is a 13 C-NMR spectrum of the compound Mangicol P of the present invention dissolved in pyridine-d 5 .
图29为本发明化合物Mangicol H溶于pyridine-d5中的HSQC谱图。Figure 29 is the HSQC spectrum of the compound Mangicol H of the present invention dissolved in pyridine-d 5 .
图30为本发明化合物Mangicol I溶于pyridine-d5中的HSQC谱图。Figure 30 is the HSQC spectrum of the compound Mangicol I of the present invention dissolved in pyridine-d 5 .
图31为本发明化合物Mangicol J溶于pyridine-d5中的HSQC谱图。Figure 31 is the HSQC spectrum of the compound Mangicol J of the present invention dissolved in pyridine-d 5 .
图32为本发明化合物Mangicol K溶于pyridine-d5中的HSQC谱图。Figure 32 is the HSQC spectrum of the compound Mangicol K of the present invention dissolved in pyridine-d 5 .
图33为本发明化合物Mangicol L溶于pyridine-d5中的HSQC谱图。Figure 33 is the HSQC spectrum of the compound Mangicol L of the present invention dissolved in pyridine-d 5 .
图34为本发明化合物Mangicol M溶于pyridine-d5中的HSQC谱图。Figure 34 is the HSQC spectrum of the compound Mangicol M of the present invention dissolved in pyridine-d 5 .
图35为本发明化合物Mangicol N溶于pyridine-d5中的HSQC谱图。Figure 35 is the HSQC spectrum of the compound Mangicol N of the present invention dissolved in pyridine-d 5 .
图36为本发明化合物Mangicol O溶于pyridine-d5中的HSQC谱图。Figure 36 is the HSQC spectrum of the compound Mangicol O of the present invention dissolved in pyridine-d 5 .
图37为本发明化合物Mangicol P溶于pyridine-d5中的HSQC谱图。Figure 37 is the HSQC spectrum of the compound Mangicol P of the present invention dissolved in pyridine-d 5 .
图38为本发明化合物Mangicol H溶于pyridine-d5中的1H-1H COSY谱图。Figure 38 is a 1 H- 1 H COZY spectrum of the compound Mangicol H of the present invention dissolved in pyridine-d 5 .
图39为本发明化合物Mangicol I溶于pyridine-d5中的1H-1H COSY谱图。Figure 39 is a 1 H- 1 H COZY spectrum of the compound Mangicol I of the present invention dissolved in pyridine-d 5 .
图40为本发明化合物Mangicol J溶于pyridine-d5中的1H-1H COSY谱图。Figure 40 is a 1 H- 1 H COSY spectrum of the compound Mangicol J of the present invention dissolved in pyridine-d 5 .
图41为本发明化合物Mangicol K溶于pyridine-d5中的1H-1H COSY谱图。Figure 41 is a 1 H- 1 H COSY spectrum of the compound Mangicol K of the present invention dissolved in pyridine-d 5 .
图42为本发明化合物Mangicol L溶于pyridine-d5中的1H-1H COSY谱图。Figure 42 is a 1 H- 1 H COSY spectrum of the compound Mangicol L of the present invention dissolved in pyridine-d 5 .
图43为本发明化合物Mangicol M溶于pyridine-d5中的1H-1H COSY谱图。Figure 43 is a 1 H- 1 H COSY spectrum of the compound Mangicol M of the present invention dissolved in pyridine-d 5 .
图44为本发明化合物Mangicol N溶于pyridine-d5中的1H-1H COSY谱图。Figure 44 is a 1 H- 1 H COSY spectrum of the compound Mangicol N of the present invention dissolved in pyridine-d 5 .
图45为本发明化合物Mangicol O溶于pyridine-d5中的1H-1H COSY谱图。Figure 45 is a 1 H- 1 H COSY spectrum of the compound Mangicol O of the present invention dissolved in pyridine-d 5 .
图46为本发明化合物Mangicol P溶于pyridine-d5中的1H-1H COSY谱图。Figure 46 is a 1 H- 1 H COSY spectrum of the compound Mangicol P of the present invention dissolved in pyridine-d 5 .
图47为本发明化合物Mangicol H溶于pyridine-d5中的HMBC谱图。Figure 47 is the HMBC spectrum of the compound Mangicol H of the present invention dissolved in pyridine-d 5 .
图48为本发明化合物Mangicol I溶于pyridine-d5中的HMBC谱图。Figure 48 is the HMBC spectrum of the compound Mangicol I of the present invention dissolved in pyridine-d 5 .
图49为本发明化合物Mangicol J溶于pyridine-d5中的HMBC谱图。Figure 49 is the HMBC spectrum of the compound Mangicol J of the present invention dissolved in pyridine-d 5 .
图50为本发明化合物Mangicol K溶于pyridine-d5中的HMBC谱图。Figure 50 is an HMBC spectrum of the compound Mangicol K of the present invention dissolved in pyridine-d 5 .
图51为本发明化合物Mangicol L溶于pyridine-d5中的HMBC谱图。Figure 51 is the HMBC spectrum of the compound Mangicol L of the present invention dissolved in pyridine-d 5 .
图52为本发明化合物Mangicol M溶于pyridine-d5中的HMBC谱图。Figure 52 is an HMBC spectrum of the compound Mangicol M of the present invention dissolved in pyridine-d 5 .
图53为本发明化合物Mangicol N溶于pyridine-d5中的HMBC谱图。Figure 53 is the HMBC spectrum of the compound Mangicol N of the present invention dissolved in pyridine-d 5 .
图54为本发明化合物Mangicol O溶于pyridine-d5中的HMBC谱图。Figure 54 is the HMBC spectrum of the compound Mangicol O of the present invention dissolved in pyridine-d 5 .
图55为本发明化合物Mangicol P溶于pyridine-d5中的HMBC谱图。Figure 55 is the HMBC spectrum of the compound Mangicol P of the present invention dissolved in pyridine-d 5 .
图56为本发明化合物Mangicol H溶于pyridine-d5中的NOESY谱图。Figure 56 is a NOESY spectrum of the compound Mangicol H of the present invention dissolved in pyridine-d 5 .
图57为本发明化合物Mangicol I溶于pyridine-d5中的NOESY谱图。Figure 57 is a NOESY spectrum of the compound Mangicol I of the present invention dissolved in pyridine-d 5 .
图58为本发明化合物Mangicol J溶于pyridine-d5中的NOESY谱图。Figure 58 is a NOESY spectrum of the compound Mangicol J of the present invention dissolved in pyridine-d 5 .
图59为本发明化合物Mangicol K溶于pyridine-d5中的NOESY谱图。Figure 59 is a NOESY spectrum of the compound Mangicol K of the present invention dissolved in pyridine-d 5 .
图60为本发明化合物Mangicol L溶于pyridine-d5中的NOESY谱图。Figure 60 is a NOESY spectrum of the compound Mangicol L of the present invention dissolved in pyridine-d 5 .
图61为本发明化合物Mangicol M溶于pyridine-d5中的NOESY谱图。Figure 61 is a NOESY spectrum of the compound Mangicol M of the present invention dissolved in pyridine-d 5 .
图62为本发明化合物Mangicol N溶于pyridine-d5中的NOESY谱图。Figure 62 is a NOESY spectrum of the compound Mangicol N of the present invention dissolved in pyridine-d 5 .
图63为本发明化合物Mangicol O溶于pyridine-d5中的NOESY谱图。Figure 63 is a NOESY spectrum of the compound Mangicol O of the present invention dissolved in pyridine-d 5 .
图64为本发明化合物Mangicol P溶于pyridine-d5中的NOESY谱图。Figure 64 is a NOESY spectrum of the compound Mangicol P of the present invention dissolved in pyridine-d 5 .
图65为本发明化合物Mangicol J的ICD图谱。Figure 65 is the ICD spectrum of the compound Mangicol J of the present invention.
具体实施方式Detailed ways
下面结合具体实施例对本发明内容进行详细说明。如下所述例子仅是本发明的较佳实施方式而已，并非对本发明作任何形式上的限制，凡是依据本发明的技术实质对实施方式所做的任何简单修改，等同变化与修饰，均属于本发明技术方案的范围内。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。The content of the present invention will be described in detail below with reference to specific embodiments. The examples described below are only preferred embodiments of the present invention and are not intended to limit the present invention in any form. Any simple modifications, equivalent changes and modifications made to the embodiments based on the technical essence of the present invention all fall within the scope of this invention. within the scope of the technical solution of the invention. The experimental methods used in the following examples are conventional methods unless otherwise specified.
本发明实施例中所采用的PCR扩增，质粒提取、转化等基础分子生物学实验技术如无特殊说明，通常按照常规方法操作，具体可参见《分子克隆实验指南》(第三版)(SambrookJ,Russell DW，Janssen K,Argentine J.黄培堂等译，2002，北京：科学出版社)，或按照相关产商提供的说明书执行。Unless otherwise specified, basic molecular biology experimental techniques such as PCR amplification, plasmid extraction, and transformation used in the examples of the present invention are usually operated according to conventional methods. For details, please refer to "Molecular Cloning Experiment Guide" (Third Edition) (SambrookJ , Russell DW, Janssen K, Argentine J. Translated by Huang Peitang et al., 2002, Beijing: Science Press), or follow the instructions provided by the relevant manufacturer.
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.
本发明所提供的四环二倍半萜类化合物Mangicols的合成基因簇，是从尖孢镰刀菌14005中克隆得到，所述基因簇含有6个基因，分别为编码二倍半萜合酶FoMS的基因fomdE或其功能等同体，编码细胞色素P450酶fomdA，fomdC，fomdD的基因fomdA，fomdC，fomdD其功能等同体，编码醛酮还原酶fomdB的基因fomdB或其功能等同体，编码水解酶fomdF的基因fomdF或其功能等同体，其中，所述fomdA的核苷酸序列如SEQ ID NO.1所示，fomdB的核苷酸序列如SEQ ID NO.2所示，fomdC的核苷酸序列如SEQ ID NO.3所示，fomdD的核苷酸序列如SEQ ID NO.4所示，fomdF的核苷酸序列如SEQ ID NO.5所示，fomdE的核苷酸序列如SEQ IDNO.6所示，或者所述基因核苷酸序列是分别与编码蛋白fomdA，fomdB，fomdC，fomdD，fomdF和FoMS的氨基酸序列一致性高于80％所对应的DNA编码序列。The synthetic gene cluster of the tetracyclic sesquiterpenoid compound Mangicols provided by the present invention is cloned from Fusarium oxysporum 14005. The gene cluster contains 6 genes, respectively encoding the sesquiterpene synthase FoMS. The gene fomdE or its functional equivalent, encoding the cytochrome P450 enzymes fomdA, fomdC, fomdD; the gene fomdA, fomdC, fomdD and its functional equivalent, encoding the aldehyde-keto reductase fomdB; the gene fomdB or its functional equivalent, encoding the hydrolase fomdF Gene fomdF or its functional equivalent, wherein the nucleotide sequence of fomdA is shown in SEQ ID NO.1, the nucleotide sequence of fomdB is shown in SEQ ID NO.2, and the nucleotide sequence of fomdC is shown in SEQ ID NO.3 is shown, the nucleotide sequence of fomdD is shown in SEQ ID NO.4, the nucleotide sequence of fomdF is shown in SEQ ID NO.5, and the nucleotide sequence of fomdE is shown in SEQ ID NO.6 , or the nucleotide sequence of the gene is a DNA coding sequence corresponding to an amino acid sequence identity of more than 80% with the coding proteins fomdA, fomdB, fomdC, fomdD, fomdF and FoMS respectively.
实施例1Example 1
一种四环二倍半萜类Mangicols的合成基因的异源表达及二倍半萜骨架化合物的结构鉴定。Heterologous expression of a synthetic gene for tetracyclic sesquiterpenes Mangicols and structural identification of sesquiterpene skeleton compounds.
利用异源表达的方法，将尖孢镰刀菌14005菌体中的Mangicol H-P生物合成基因簇通过构建表达质粒转入宿主米曲霉中，检测异源表达菌株产物生产情况。本实施例所用到培养基配方如表1。Using the heterologous expression method, the Mangicol H-P biosynthetic gene cluster in Fusarium oxysporum 14005 cells was constructed and transferred into the host Aspergillus oryzae by constructing an expression plasmid, and the production of the heterologous expression strain product was detected. The culture medium formula used in this example is shown in Table 1.
表1实施例所用到培养基配方Table 1 Examples of media formulations used
1、Mangicols H-P基因簇异源表达载体的构建1. Construction of Mangicols H-P gene cluster heterologous expression vector
(1)以F.oxysporum 14005的基因组为模板，分别以引物fomdE-F/fomdE-R，fomdA-F/fomdA-R，fomdC-F/fomdC-R，fomdD-F/fomdD-R，fomdB-F/fomdB-R和fomdF-F/fomdF-R对二倍半萜合酶的基因fomdE，细胞色素P450酶基因fomdA，fomdC，fomdD，醛酮还原酶基因fomdB，水解酶基因fomdF，进行PCR扩增。(1) Using the genome of F. oxysporum 14005 as a template, use primers fomdE-F/fomdE-R, fomdA-F/fomdA-R, fomdC-F/fomdC-R, fomdD-F/fomdD-R, fomdB- respectively. F/fomdB-R and fomdF-F/fomdF-R performed PCR amplification of the bisquiterpene synthase gene fomdE, cytochrome P450 enzyme genes fomdA, fomdC, fomdD, aldehyde-keto reductase gene fomdB, and hydrolase gene fomdF. increase.
(2)基因fomdE，fomdA和fomdC的PCR产物经核酸纯化试剂盒纯化后，再利用Ezmax重组试剂盒将fomdE整合至KpnI酶切消化后的线性载体pUARA4中，连接产物转化到大肠杆菌DH10B中，通过氨苄青霉素筛选阳性转化子。液体培养阳性转化子，提取质粒PCR验证，获取pUARA4-fomdE质粒；在此基础上，再利用Ezmax重组试剂盒将fomdA整合至PacI酶切消化后的线性载体pUARA4-fomdE，连接产物转化到大肠杆菌DH10B中，通过氨苄青霉素筛选阳性转化子。液体培养阳性转化子，提取质粒PCR验证，获取pUARA4-fomdAE质粒；最后利用Ezmax重组试剂盒将fomdC整合至NheI酶切消化后的线性载体pUARA4-fomdAE，连接产物转化到大肠杆菌DH10B中，通过氨苄青霉素筛选阳性转化子。液体培养阳性转化子，提取质粒PCR验证，获取表达载体pUARA4-fomdACE质粒。(2) After the PCR products of genes fomdE, fomdA and fomdC are purified with a nucleic acid purification kit, the Ezmax recombination kit is used to integrate fomdE into the linear vector pUARA4 digested by KpnI enzyme, and the ligation product is transformed into E. coli DH10B. Positive transformants were selected by ampicillin. The positive transformants were cultured in liquid, the plasmid was extracted and verified by PCR, and the pUARA4-fomdE plasmid was obtained; on this basis, the Ezmax recombination kit was used to integrate fomdA into the PacI-digested linear vector pUARA4-fomdE, and the ligated product was transformed into E. coli In DH10B, positive transformants were selected by ampicillin. The positive transformants were cultured in liquid, the plasmid was extracted and verified by PCR, and the pUARA4-fomdAE plasmid was obtained; finally, the Ezmax recombination kit was used to integrate fomdC into the linear vector pUARA4-fomdAE digested by NheI, and the ligated product was transformed into E. coli DH10B and passed through ampicillin. Penicillin was used to screen positive transformants. The positive transformants were cultured in liquid, the plasmid was extracted and verified by PCR, and the expression vector pUARA4-fomdACE plasmid was obtained.
(3)同样，以如上方法在基因fomdD，fomdB和fomdF的PCR产物经核酸纯化试剂盒纯化后，再利用Ezmax重组试剂盒将fomdD整合至KpnI酶切消化后的线性载体pUSA4中，连接产物转化到大肠杆菌DH10B中，通过氨苄青霉素筛选阳性转化子。液体培养阳性转化子，提取质粒PCR验证，获取pUSA4-fomdD质粒；在此基础上，再利用Ezmax重组试剂盒将fomdB整合至PacI酶切消化后的线性载体pUSA4-fomdD，连接产物转化到大肠杆菌DH10B中，通过氨苄青霉素筛选阳性转化子。液体培养阳性转化子，提取质粒PCR验证，获取pUSA4-fomdBD质粒；最后利用Ezmax重组试剂盒将fomdF整合至NheI酶切消化后的线性载体pUSA4-fomdBD，连接产物转化到大肠杆菌DH10B中，通过氨苄青霉素筛选阳性转化子。液体培养阳性转化子，提取质粒PCR验证，获取表达载体pUSA4-fomdBDF质粒。(3) Similarly, after the PCR products of genes fomdD, fomdB and fomdF are purified by the nucleic acid purification kit in the same way as above, the Ezmax recombination kit is used to integrate fomdD into the linear vector pUSA4 digested by KpnI, and the ligated product is transformed into E. coli DH10B, and positive transformants were selected by ampicillin. The positive transformants were cultured in liquid, the plasmid was extracted and verified by PCR, and the pUSA4-fomdD plasmid was obtained; on this basis, the Ezmax recombination kit was used to integrate fomdB into the linear vector pUSA4-fomdD digested by PacI, and the ligated product was transformed into E. coli In DH10B, positive transformants were selected by ampicillin. The positive transformants were cultured in liquid, the plasmid was extracted and verified by PCR, and the pUSA4-fomdBD plasmid was obtained; finally, the Ezmax recombination kit was used to integrate fomdF into the linear vector pUSA4-fomdBD digested by NheI, and the ligated product was transformed into E. coli DH10B and passed through ampicillin. Penicillin was used to screen positive transformants. The positive transformants were cultured in liquid, the plasmid was extracted and verified by PCR, and the expression vector pUSA4-fomdBDF plasmid was obtained.
表2实例中用到的引物序列Primer sequences used in the examples in Table 2
2、原生质体的转化2. Transformation of protoplasts
(1)将米曲霉A.oryzae NSAR1涂于PDA平板，30℃培养7天。(1) Spread Aspergillus oryzae NSAR1 on a PDA plate and culture it at 30°C for 7 days.
(2)收集抱子于10mL 0.1％Tween-80(一般需要收集1个平板的抱子)，用血球计数板计数。接种约107个孢子于50mL DPY中，30℃、220rpm培养2-3天。(2) Collect spores in 10 mL of 0.1% Tween-80 (generally one plate of spores needs to be collected) and count with a hemocytometer. Inoculate about 10 7 spores into 50mL DPY and culture at 30°C and 220rpm for 2-3 days.
(3)称量100mg Yatalase，加入solution 0溶解，20mL用0.22μm滤膜过滤灭菌，加入到50mL离心管中。(3) Weigh 100 mg Yatalase, add solution 0 to dissolve, filter and sterilize 20 mL with a 0.22 μm filter membrane, and add it to a 50 mL centrifuge tube.
(4)收集菌体。将培养好的100mL菌丝体倒入到P250玻璃过滤器中，去除培养基，用灭菌水清洗(或0.8M NaCl)3～5次，用灭菌药勺将水分挤干去除，然后将压干的菌体加入到Yatalase溶液中。30℃，200rpm震荡培养1-2小时，至球状菌丝体消失上清明显秽浊状为止。(4) Collect bacterial cells. Pour 100mL of cultured mycelium into a P250 glass filter, remove the culture medium, wash with sterile water (or 0.8M NaCl) 3 to 5 times, squeeze out the water with a sterile spoon, and then The dried bacterial cells were added to the Yatalase solution. Culture at 30°C with shaking at 200rpm for 1-2 hours until the spherical mycelium disappears and the supernatant becomes obviously turbid.
(5)用Miracloth滤布过滤消化好的菌液，收集原生质体，转到新50mL离心管中，4℃，800g，5min离心。(5) Use Miracloth filter cloth to filter the digested bacterial solution, collect the protoplasts, transfer them to a new 50mL centrifuge tube, and centrifuge at 800g and 4°C for 5 minutes.
(6)去除上清液，加入20mL，0.8M NaCl再悬浮清洗，4℃，800g，5min离心(清洗两次)。去除上清液，加10mL，0.8M NaCl。用细菌计数器在显微镜下数原生质体的数目。原生质体数目＝总计数/80x 400ml x104 x稀释倍数。(6) Remove the supernatant, add 20 mL of 0.8M NaCl, resuspend and wash, centrifuge at 4°C, 800g, 5 min (wash twice). Remove the supernatant and add 10 mL of 0.8M NaCl. Use a bacterial counter to count the number of protoplasts under a microscope. Number of protoplasts = total count/80x 400ml x10 4 x dilution factor.
(7)将原生质体浓度调成2x 108cell/mL。(sol 2/sol 3＝4/1)，依据菌体生长情况可收获0.5mL-2mL原生质体不等。(7) Adjust the protoplast concentration to 2x 10 8 cell/mL. (sol 2/sol 3=4/1), depending on the bacterial growth, 0.5mL-2mL protoplasts can be harvested.
(8)取200μL的原生质体溶液移至新的50mL的离心管中，分别加入10μg表达质粒pUARA4-fomdACE和pUSA4-fomdBDF，轻轻地混匀。在冰上静置20min。期间将灭菌好的Topagar，在50℃水浴中保温。(8) Transfer 200 μL of the protoplast solution to a new 50 mL centrifuge tube, add 10 μg of expression plasmids pUARA4-fomdACE and pUSA4-fomdBDF respectively, and mix gently. Let stand on ice for 20min. During this period, the sterilized Topagar was kept warm in a 50°C water bath.
(9)往10的混悬液中加入1mL的sol 3，用枪头轻轻的混匀。在室温下静置20min。加入10mL的sol 2，轻轻混匀。(9) Add 1 mL of sol 3 to the suspension of 10, and mix gently with a pipette tip. Let stand at room temperature for 20 minutes. Add 10 mL of sol 2 and mix gently.
(10)4℃，800g，10min离心，除去上清，加入1mL的sol 2，用移液枪轻轻地混悬，加入200μL到固体筛选培养基的中央(x3板)。在培养皿的周围迅速加入5mL 50℃保温的topagar，快速混匀。平板表面充分干燥后，用parafilm缠好，盖朝下，进行30℃培养3-7天。(10) Centrifuge at 800 g for 10 min at 4°C, remove the supernatant, add 1 mL of sol 2, gently suspend with a pipette, and add 200 μL to the center of the solid screening medium (x3 plate). Quickly add 5 mL of topagar insulated at 50°C around the petri dish and mix quickly. After the surface of the plate is fully dry, wrap it with parafilm, with the cover facing down, and culture it at 30°C for 3-7 days.
(11)每平板挑2-3个克隆，共8个。将长出的转化子进行PCR验证，阳性转化子即为Mangicols H-P基因簇异源表达菌株AO-fomdABCDEF。(11) Pick 2-3 clones per plate, totaling 8 clones. The grown transformants were verified by PCR, and the positive transformants were Mangicols H-P gene cluster heterologous expression strain AO-fomdABCDEF.
3、异源表达菌株AO-fomdABCDEF表达产物的分离纯化3. Isolation and purification of the expression product of heterologous expression strain AO-fomdABCDEF
接种异源表达菌株AO-fomdABCDEF菌丝体接种于含0.1％腺嘌呤的MPY培养基中，于30℃，220rpm培养2天作为种子液，按照80g大米，120mL去离子水加5mL种子液的比例，接种于添加千分之一腺嘌呤的大米固体培养基中，30℃，静置培养18天。Inoculate the mycelium of the heterologous expression strain AO-fomdABCDEF into MPY medium containing 0.1% adenine, and culture it at 30°C and 220rpm for 2 days as a seed liquid. According to the ratio of 80g rice, 120mL deionized water and 5mL seed liquid , inoculated into rice solid medium supplemented with one thousandth of adenine, and cultured statically at 30°C for 18 days.
将AO-fomdABCDEF的固体发酵物加入等体积的乙酸乙酯萃取3次，蒸干后获得浸膏。浸膏用石油醚萃取获得极性小的组分，石油醚萃取液蒸干后进行正相分离，以石油醚和乙酸乙酯为流动相进行洗脱并富集目标组分Fr.5和Fr.7。Fr.5进行凝胶柱分离，最终用反相Cholester色谱柱，乙腈/0.1％甲酸水体积比80:20为流动相洗脱获得Mangicol K。Fr.7进行凝胶柱分离获得三个馏分Fr.7.1-Fr.7.3.Fr.7.2用ACE C18-PFP色谱柱进行半制备，以乙腈/0.1％甲酸水体积比75:25为流动相洗脱获得Mangicol O和馏分Fr.7.2.1；馏分Fr.7.2.1进一步用手型柱Chiralpak IA，以正己烷/乙醇体积比95：5为流动相获得Mangicols H和I。Fr.7.3用Phenomenex色谱柱进行半制备，以乙腈/0.1％甲酸水体积比80:20为流动相洗脱获得Mangicols L,N和P以及馏分Fr.7.3.1；馏分Fr.7.3.1进一步用手型柱Chiralpak IA，以正己烷/乙醇体积比94：6为流动相获得Mangicols J和M。Add the solid fermentation product of AO-fomdABCDEF to an equal volume of ethyl acetate for extraction three times, and then evaporate to dryness to obtain the extract. The extract is extracted with petroleum ether to obtain small polar components. The petroleum ether extract is evaporated to dryness and then subjected to normal phase separation. Petroleum ether and ethyl acetate are used as mobile phases to elute and enrich the target components Fr.5 and Fr. .7. Fr.5 was used for gel column separation, and finally a reversed-phase Cholester chromatography column was used to elute with acetonitrile/0.1% formic acid and water volume ratio of 80:20 to obtain Mangicol K. Fr.7 was subjected to gel column separation to obtain three fractions Fr.7.1-Fr.7.3.Fr.7.2, which were semi-prepared using an ACE C18-PFP chromatographic column and washed with acetonitrile/0.1% formic acid and water volume ratio of 75:25 as the mobile phase. Mangicol O and fraction Fr.7.2.1 were obtained by removal; fraction Fr.7.2.1 was further processed with a hand-type column Chiralpak IA, using n-hexane/ethanol volume ratio 95:5 as the mobile phase to obtain Mangicols H and I. Fr.7.3 was semi-prepared using a Phenomenex chromatography column, and the mobile phase was eluted with acetonitrile/0.1% formic acid water volume ratio of 80:20 to obtain Mangicols L, N and P as well as the fraction Fr.7.3.1; the fraction Fr.7.3.1 was further Mangicols J and M were obtained using Chiralpak IA, a hand-type column, with n-hexane/ethanol volume ratio 94:6 as the mobile phase.
分离的四环二倍半萜类的NMR测试采用Bruker 600MHz(1H 600MHz；13C 150MHz)，溶剂为氘代吡啶。The NMR test of the isolated tetracyclic sesquiterpenes was carried out using Bruker 600MHz ( 1 H 600MHz; 13 C 150MHz), and the solvent was deuterated pyridine.
4、鉴定四环二倍半萜类Mangicols H-P。4. Identification of tetracyclic bisquiterpenes Mangicols H-P.
将上述得到的四环二倍半萜类Mangicols H-P进行鉴定：Identification of the tetracyclic bisquiterpene Mangicols H-P obtained above:
(1)外观：为淡黄色透明油状。(1) Appearance: light yellow transparent oil.
(2)溶解性：易溶于甲醇，难溶于水。(2) Solubility: Easily soluble in methanol, difficult to dissolve in water.
(3)紫外光谱：化合物Mangicols H-P甲醇溶液的紫外光谱在210nm处有最大吸收峰，紫外光谱见图1所示，图1为本发明化合物Mangicols H-P的紫外谱图。紫外光谱测试仪器为Mariner System 5304instrument。(3) Ultraviolet spectrum: The ultraviolet spectrum of the methanol solution of compound Mangicols H-P has a maximum absorption peak at 210 nm. The ultraviolet spectrum is shown in Figure 1. Figure 1 is the ultraviolet spectrum of the compound Mangicols H-P of the present invention. The UV spectrum testing instrument is Mariner System 5304 instrument.
(4)质谱：图2为本发明化合物Mangicol H的HR-ESI-MS谱图，显示其[M+H]+峰为m/z 357.31519，提示其最可能分子式为C25H40O。图3为本发明化合物Mangicol I的HR-ESI-MS谱图，显示其[M+H]+峰为m/z 357.31519，提示其最可能分子式为C25H40O。图4为本发明化合物Mangicol J的HR-ESI-MS谱图，显示其[M-H2O+H]+峰为m/z371.29501，提示其最可能分子式为C25H39O2。图5为本发明化合物Mangicol K的HR-ESI-MS谱图，显示其[M+H]+峰为m/z387.28937，提示其最可能分子式为C25H38O3。图6为本发明化合物Mangicol L的HR-ESI-MS谱图，显示其[M+H]+峰为m/z 389.30502，提示其最可能分子式为C25H40O3。图7为本发明化合物Mangicol M的HR-ESI-MS谱图，显示其[M-H2O+H]+峰为m/z 371.29501，提示其最可能分子式为C25H39O2。图8为本发明化合物Mangicol N的HR-ESI-MS谱图，显示其[M-H2O+H]+峰为m/z389.30557，提示其最可能分子式为C25H41O3。图9为本发明化合物Mangicol O的HR-ESI-MS谱图，显示其[M-H2O+H]+峰为m/z 431.31494，提示其最可能分子式为C27H45O5。图10为本发明化合物Mangicol P的HR-ESI-MS谱图，显示其[M-H2O+H]+峰为m/z 507.34674，提示其最可能分子式为C33H49O5。HR-ESI-MS图谱测试采用Thermal Fisher Orbitrap Q Exactive massspectrometer，甲醇为溶剂。(4) Mass spectrum: Figure 2 is the HR-ESI-MS spectrum of the compound Mangicol H of the present invention, showing that its [M+H] + peak is m/z 357.31519, suggesting that its most likely molecular formula is C 25 H 40 O. Figure 3 is the HR-ESI-MS spectrum of the compound Mangicol I of the present invention, showing that its [M+H] + peak is m/z 357.31519, suggesting that its most likely molecular formula is C 25 H 40 O. Figure 4 is the HR-ESI-MS spectrum of the compound Mangicol J of the present invention, showing that its [MH 2 O+H] + peak is m/z371.29501, suggesting that its most likely molecular formula is C 25 H 39 O 2 . Figure 5 is the HR-ESI-MS spectrum of the compound Mangicol K of the present invention, showing that its [M+H] + peak is m/z387.28937, suggesting that its most likely molecular formula is C 25 H 38 O 3 . Figure 6 is the HR-ESI-MS spectrum of the compound Mangicol L of the present invention, showing that its [M+H] + peak is m/z 389.30502, suggesting that its most likely molecular formula is C 25 H 40 O 3 . Figure 7 is the HR-ESI-MS spectrum of the compound Mangicol M of the present invention, showing that its [MH 2 O+H] + peak is m/z 371.29501, suggesting that its most likely molecular formula is C 25 H 39 O 2 . Figure 8 is the HR-ESI-MS spectrum of the compound Mangicol N of the present invention, showing that its [MH 2 O+H] + peak is m/z389.30557, suggesting that its most likely molecular formula is C 25 H 41 O 3 . Figure 9 is the HR-ESI-MS spectrum of the compound Mangicol O of the present invention, showing that its [MH 2 O+H] + peak is m/z 431.31494, suggesting that its most likely molecular formula is C 27 H 45 O 5 . Figure 10 is the HR-ESI-MS spectrum of the compound Mangicol P of the present invention, showing that its [MH 2 O+H] + peak is m/z 507.34674, suggesting that its most likely molecular formula is C 33 H 49 O 5 . The HR-ESI-MS spectrum was tested using a Thermal Fisher Orbitrap Q Exactive massspectrometer, with methanol as the solvent.
(5)核磁共振谱：图11为本发明化合物Mangicol H溶于pyridine-d5中的1H-NMR谱图。图12为本发明化合物Mangicol I溶于pyridine-d5中的1H-NMR谱图。图13为本发明化合物Mangicol J溶于pyridine-d5中的1H-NMR谱图。图14为本发明化合物Mangicol K溶于pyridine-d5中的1H-NMR谱图。图15为本发明化合物Mangicol L溶于pyridine-d5中的1H-NMR谱图。图16为本发明化合物Mangicol M溶于pyridine-d5中的1H-NMR谱图。图17为本发明化合物Mangicol N溶于pyridine-d5中的1H-NMR谱图。图18为本发明化合物Mangicol O溶于pyridine-d5中的1H-NMR谱图。图19为本发明化合物Mangicol P溶于pyridine-d5中的1H-NMR谱图。图20为本发明化合物Mangicol H溶于pyridine-d5中的13C-NMR谱图。图21为本发明化合物Mangicol I溶于pyridine-d5中的13C-NMR谱图。图22为本发明化合物Mangicol J溶于pyridine-d5中的13C-NMR谱图。图23为本发明化合物Mangicol K溶于pyridine-d5中的13C-NMR谱图。图24为本发明化合物Mangicol L溶于pyridine-d5中的13C-NMR谱图。图25为本发明化合物Mangicol M溶于pyridine-d5中的13C-NMR谱图。图26为本发明化合物Mangicol N溶于pyridine-d5中的13C-NMR谱图。图27为本发明化合物Mangicol O溶于pyridine-d5中的13C-NMR谱图。图28为本发明化合物Mangicol P溶于pyridine-d5中的13C-NMR谱图。图29为本发明化合物Mangicol H溶于pyridine-d5中的HSQC谱图。图30为本发明化合物Mangicol I溶于pyridine-d5中的HSQC谱图。图31为本发明化合物Mangicol J溶于pyridine-d5中的HSQC谱图。图32为本发明化合物Mangicol K溶于pyridine-d5中的HSQC谱图。图33为本发明化合物Mangicol L溶于pyridine-d5中的HSQC谱图。图34为本发明化合物Mangicol M溶于pyridine-d5中的HSQC谱图。图35为本发明化合物Mangicol N溶于pyridine-d5中的HSQC谱图。图36为本发明化合物Mangicol O溶于pyridine-d5中的HSQC谱图。图37为本发明化合物Mangicol P溶于pyridine-d5中的HSQC谱图。图38为本发明化合物Mangicol H溶于pyridine-d5中的1H-1H COSY谱图。图39为本发明化合物Mangicol I溶于pyridine-d5中的1H-1H COSY谱图。图40为本发明化合物Mangicol J溶于pyridine-d5中的1H-1H COSY谱图。图41为本发明化合物Mangicol K溶于pyridine-d5中的1H-1H COSY谱图。图42为本发明化合物Mangicol L溶于pyridine-d5中的1H-1H COSY谱图。图43本发明化合物Mangicol M溶于pyridine-d5中的1H-1H COSY谱图。图44为本发明化合物Mangicol N溶于pyridine-d5中的1H-1H COSY谱图。图45为本发明化合物Mangicol O溶于pyridine-d5中的1H-1H COSY谱图。图46为本发明化合物Mangicol P溶于pyridine-d5中的1H-1H COSY谱图。图47为本发明化合物Mangicol H溶于pyridine-d5中的HMBC谱图。图48为本发明化合物Mangicol I溶于pyridine-d5中的HMBC谱图。图49为本发明化合物Mangicol J溶于pyridine-d5中的HMBC谱图。图50为本发明化合物Mangicol K溶于pyridine-d5中的HMBC谱图。图51为本发明化合物Mangicol L溶于pyridine-d5中的HMBC谱图。图52为本发明化合物Mangicol M溶于pyridine-d5中的HMBC谱图。图53为本发明化合物Mangicol N溶于pyridine-d5中的HMBC谱图。图54为本发明化合物Mangicol O溶于pyridine-d5中的HMBC谱图。图55为本发明化合物Mangicol P溶于pyridine-d5中的HMBC谱图。图56为本发明化合物Mangicol H溶于pyridine-d5中的NOESY谱图。图57为本发明化合物Mangicol I溶于pyridine-d5中的NOESY谱图。图58为本发明化合物Mangicol J溶于pyridine-d5中的NOESY谱图。图59为本发明化合物Mangicol K溶于pyridine-d5中的NOESY谱图。图60为本发明化合物Mangicol L溶于pyridine-d5中的NOESY谱图。图61为本发明化合物Mangicol M溶于pyridine-d5中的NOESY谱图。图62为本发明化合物Mangicol N溶于pyridine-d5中的NOESY谱图。图63为本发明化合物Mangicol O溶于pyridine-d5中的NOESY谱图。图64为本发明化合物Mangicol P溶于pyridine-d5中的NOESY谱图。(5) Nuclear Magnetic Resonance Spectrum: Figure 11 is a 1 H-NMR spectrum of the compound Mangicol H of the present invention dissolved in pyridine-d 5 . Figure 12 is a 1 H-NMR spectrum of the compound Mangicol I of the present invention dissolved in pyridine-d 5 . Figure 13 is a 1 H-NMR spectrum of the compound Mangicol J of the present invention dissolved in pyridine-d 5 . Figure 14 is a 1 H-NMR spectrum of the compound Mangicol K of the present invention dissolved in pyridine-d 5 . Figure 15 is a 1 H-NMR spectrum of the compound Mangicol L of the present invention dissolved in pyridine-d 5 . Figure 16 is a 1 H-NMR spectrum of the compound Mangicol M of the present invention dissolved in pyridine-d 5 . Figure 17 is a 1 H-NMR spectrum of the compound Mangicol N of the present invention dissolved in pyridine-d 5 . Figure 18 is a 1 H-NMR spectrum of the compound Mangicol O of the present invention dissolved in pyridine-d 5 . Figure 19 is a 1 H-NMR spectrum of the compound Mangicol P of the present invention dissolved in pyridine-d 5 . Figure 20 is a 13 C-NMR spectrum of the compound Mangicol H of the present invention dissolved in pyridine-d 5 . Figure 21 is a 13 C-NMR spectrum of the compound Mangicol I of the present invention dissolved in pyridine-d 5 . Figure 22 is a 13 C-NMR spectrum of the compound Mangicol J of the present invention dissolved in pyridine-d 5 . Figure 23 is a 13 C-NMR spectrum of the compound Mangicol K of the present invention dissolved in pyridine-d 5 . Figure 24 is a 13 C-NMR spectrum of the compound Mangicol L of the present invention dissolved in pyridine-d 5 . Figure 25 is a 13 C-NMR spectrum of the compound Mangicol M of the present invention dissolved in pyridine-d 5 . Figure 26 is a 13 C-NMR spectrum of the compound Mangicol N of the present invention dissolved in pyridine-d 5 . Figure 27 is a 13 C-NMR spectrum of the compound Mangicol O of the present invention dissolved in pyridine-d 5 . Figure 28 is a 13 C-NMR spectrum of the compound Mangicol P of the present invention dissolved in pyridine-d 5 . Figure 29 is the HSQC spectrum of the compound Mangicol H of the present invention dissolved in pyridine-d 5 . Figure 30 is the HSQC spectrum of the compound Mangicol I of the present invention dissolved in pyridine-d 5 . Figure 31 is the HSQC spectrum of the compound Mangicol J of the present invention dissolved in pyridine-d 5 . Figure 32 is the HSQC spectrum of the compound Mangicol K of the present invention dissolved in pyridine-d 5 . Figure 33 is the HSQC spectrum of the compound Mangicol L of the present invention dissolved in pyridine-d 5 . Figure 34 is the HSQC spectrum of the compound Mangicol M of the present invention dissolved in pyridine-d 5 . Figure 35 is the HSQC spectrum of the compound Mangicol N of the present invention dissolved in pyridine-d 5 . Figure 36 is the HSQC spectrum of the compound Mangicol O of the present invention dissolved in pyridine-d 5 . Figure 37 is the HSQC spectrum of the compound Mangicol P of the present invention dissolved in pyridine-d 5 . Figure 38 is a 1 H- 1 H COZY spectrum of the compound Mangicol H of the present invention dissolved in pyridine-d 5 . Figure 39 is a 1 H- 1 H COZY spectrum of the compound Mangicol I of the present invention dissolved in pyridine-d 5 . Figure 40 is a 1 H- 1 H COSY spectrum of the compound Mangicol J of the present invention dissolved in pyridine-d 5 . Figure 41 is a 1 H- 1 H COSY spectrum of the compound Mangicol K of the present invention dissolved in pyridine-d 5 . Figure 42 is a 1 H- 1 H COSY spectrum of the compound Mangicol L of the present invention dissolved in pyridine-d 5 . Figure 43 1 H- 1 H COZY spectrum of the compound Mangicol M of the present invention dissolved in pyridine-d 5 . Figure 44 is a 1 H- 1 H COSY spectrum of the compound Mangicol N of the present invention dissolved in pyridine-d 5 . Figure 45 is a 1 H- 1 H COSY spectrum of the compound Mangicol O of the present invention dissolved in pyridine-d 5 . Figure 46 is a 1 H- 1 H COSY spectrum of the compound Mangicol P of the present invention dissolved in pyridine-d 5 . Figure 47 is the HMBC spectrum of the compound Mangicol H of the present invention dissolved in pyridine-d 5 . Figure 48 is the HMBC spectrum of the compound Mangicol I of the present invention dissolved in pyridine-d 5 . Figure 49 is the HMBC spectrum of the compound Mangicol J of the present invention dissolved in pyridine-d 5 . Figure 50 is an HMBC spectrum of the compound Mangicol K of the present invention dissolved in pyridine-d 5 . Figure 51 is the HMBC spectrum of the compound Mangicol L of the present invention dissolved in pyridine-d 5 . Figure 52 is an HMBC spectrum of the compound Mangicol M of the present invention dissolved in pyridine-d 5 . Figure 53 is the HMBC spectrum of the compound Mangicol N of the present invention dissolved in pyridine-d 5 . Figure 54 is the HMBC spectrum of the compound Mangicol O of the present invention dissolved in pyridine-d 5 . Figure 55 is the HMBC spectrum of the compound Mangicol P of the present invention dissolved in pyridine-d 5 . Figure 56 is a NOESY spectrum of the compound Mangicol H of the present invention dissolved in pyridine-d 5 . Figure 57 is a NOESY spectrum of the compound Mangicol I of the present invention dissolved in pyridine-d 5 . Figure 58 is a NOESY spectrum of the compound Mangicol J of the present invention dissolved in pyridine-d 5 . Figure 59 is a NOESY spectrum of the compound Mangicol K of the present invention dissolved in pyridine-d 5 . Figure 60 is a NOESY spectrum of the compound Mangicol L of the present invention dissolved in pyridine-d 5 . Figure 61 is a NOESY spectrum of the compound Mangicol M of the present invention dissolved in pyridine-d 5 . Figure 62 is a NOESY spectrum of the compound Mangicol N of the present invention dissolved in pyridine-d 5 . Figure 63 is a NOESY spectrum of the compound Mangicol O of the present invention dissolved in pyridine-d 5 . Figure 64 is a NOESY spectrum of the compound Mangicol P of the present invention dissolved in pyridine-d 5 .
(6)图65为本发明化合物Mangicol J的C-19位绝对构型根据Snatzke法的ICD图谱。(6) Figure 65 is the ICD spectrum of the C-19 absolute configuration of the compound Mangicol J of the present invention according to the Snatzke method.
最终确定结构式如下：The final structural formula is as follows:
表3化合物Mangicols H-J的1H和13C-NMR谱各峰归属Table 3 The peak assignments of 1 H and 13 C-NMR spectra of compound Mangicols HJ
表4化合物Mangicols K-M的1H和13C-NMR谱各峰归属Table 4 The peak assignments of 1 H and 13 C-NMR spectra of compound Mangicols KM
表5化合物Mangicols N-P的1H和13C-NMR谱各峰归属Table 5 The peak assignments of 1 H and 13 C-NMR spectra of compound Mangicols NP
化合物Mangicols H-P的NMR测试采用Bruker 600MHz(1H 600MHz；13C 150MHz)。化合物Mangicols H-P的溶剂为pyridine-d5。The NMR test of compound Mangicols HP was carried out using Bruker 600MHz ( 1 H 600MHz; 13 C 150MHz). The solvent for compound Mangicols HP is pyridine-d 5 .
Mangicols类二倍半萜类化合物抗菌活性测试Test of Antibacterial Activity of Mangicols Disquiterpenoids
(1)对枯草芽孢杆菌、金黄色葡萄球菌、表皮葡萄球菌，铜绿假单胞菌和变形链球菌的抗菌测定(1) Antibacterial determination of Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Streptococcus mutans
所测菌株包括：枯草芽孢杆菌菌株HD11，金黄色葡萄球菌菌株ATCC 6538，表皮葡萄球菌菌株CGMCC1.1757.，铜绿假单胞菌菌株PA01和变形链球菌菌株ATCC UA159。采用连续稀释法测定化合物对所选细菌生长的抑制作用，获得化合物对抗不同菌株的最低抑菌浓度。最低抑菌浓度(Minimal inhibitory concentration,MIC)是抑制细菌生长所需药物的最低浓度。本实验选取万古霉素作为阳性对照药。The strains tested included: Bacillus subtilis strain HD11, Staphylococcus aureus strain ATCC 6538, Staphylococcus epidermidis strain CGMCC1.1757., Pseudomonas aeruginosa strain PA01 and Streptococcus mutans strain ATCC UA159. The serial dilution method was used to determine the inhibitory effect of the compound on the growth of selected bacteria, and the minimum inhibitory concentration of the compound against different strains was obtained. Minimal inhibitory concentration (MIC) is the lowest concentration of drugs required to inhibit bacterial growth. Vancomycin was selected as the positive control drug in this experiment.
测试用细菌先于Mueller-Hinton Broth(MHB)培养基培养至对数期，将培养好的细菌用培养基稀释成104cfu/mL浓度接种于96孔细胞培养板中，每孔中包含78μL细菌悬浮液，每种化合物的2倍系列稀释液添加2μL。样品和对照药溶解于二甲基亚砜(DMSO)中，DMSO的最终浓度不得高于0.05％。采用连续稀释法获得一系列样品浓度为4000至31.3μg/mL，37℃需氧条件下温育16小时。用酶标仪在600nm测培养前后吸光度，根据吸光度的变化计算得到最低抑菌浓度即MIC值，所有实验平行进行3次。The bacteria for testing were first cultured in Mueller-Hinton Broth (MHB) medium to the logarithmic phase. The cultured bacteria were diluted with the culture medium to a concentration of 10 4 cfu/mL and inoculated into a 96-well cell culture plate. Each well contained 78 μL. For bacterial suspensions, add 2 μL of 2-fold serial dilutions of each compound. Samples and control drugs were dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO should not be higher than 0.05%. A series of sample concentrations ranging from 4000 to 31.3 μg/mL were obtained using the serial dilution method and incubated under aerobic conditions at 37°C for 16 hours. Use a microplate reader to measure the absorbance before and after incubation at 600 nm, and calculate the minimum inhibitory concentration, or MIC value, based on the change in absorbance. All experiments were conducted in parallel three times.
实验结果如表7所示：The experimental results are shown in Table 7:
表7化合物Mangicols H-P的体外抗细菌活性测试结果Table 7 In vitro antibacterial activity test results of compound Mangicols H-P
体外抗细菌活性研究表明，化合物J对变形链球菌具有很强的抑制活性(MIC＝6.25μg/mL)，化合物L和M对变形链球菌也具有一定的抑制活性(MIC＝12.5μg/mL)。In vitro antibacterial activity studies show that compound J has strong inhibitory activity against Streptococcus mutans (MIC=6.25μg/mL), and compounds L and M also have certain inhibitory activity against Streptococcus mutans (MIC=12.5μg/mL) .
(2)抗真菌测定(2) Antifungal assay
所测菌株包括：白色念珠菌菌株SC 5314。采用连续稀释法测定化合物对所选细菌生长的抑制作用，获得化合物对抗不同菌株的最低抑菌浓度。最低抑菌浓度(Minimalinhibitory concentration,MIC)是抑制细菌生长所需药物的最低浓度。本实验选取两性霉素B作为阳性对照药，同时设置只加DMSO处理作为阴性对照。The strains tested included: Candida albicans strain SC 5314. The serial dilution method was used to determine the inhibitory effect of the compound on the growth of selected bacteria, and the minimum inhibitory concentration of the compound against different strains was obtained. Minimal inhibitory concentration (MIC) is the lowest concentration of drugs required to inhibit bacterial growth. In this experiment, amphotericin B was selected as the positive control drug, and only DMSO was added as the negative control.
挑取白色念珠菌菌株SC 5314单菌落悬浮在RPMI 1640中，浓度为1×104cfu/mL接种于96孔细胞培养板中，每孔中包含78μL真菌悬浮液，每种化合物的2倍系列稀释液添加2μL。样品和对照药溶解于二甲基亚砜(DMSO)中，DMSO的最终浓度不得高于0.05％。采用连续稀释法获得一系列样品浓度为4000至31.3μg/mL，并将板在35℃下孵育16小时。用酶标仪在600nm测培养前后吸光度，根据吸光度的变化计算得到最低抑菌浓度即MIC值，所有实验平行进行3次。Pick a single colony of Candida albicans strain SC 5314 and suspend it in RPMI 1640 at a concentration of 1×10 4 cfu/mL and inoculate it into a 96-well cell culture plate. Each well contains 78 μL of fungal suspension and a 2-fold series of each compound. Add 2 μL of diluent. Samples and control drugs were dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO should not be higher than 0.05%. Serial dilutions were used to obtain a range of sample concentrations from 4000 to 31.3 μg/mL, and the plates were incubated at 35°C for 16 hours. Use a microplate reader to measure the absorbance before and after incubation at 600 nm, and calculate the minimum inhibitory concentration, or MIC value, based on the change in absorbance. All experiments were conducted in parallel three times.
实验结果如表8所示：The experimental results are shown in Table 8:
表8化合物Mangicols H-P的体外抗真菌活性测试结果Table 8 In vitro antifungal activity test results of compound Mangicols H-P
以DMSO和两性霉素B为对照，以上化合物对白色念珠菌没有明显的抑制作用(MIC值为≥50mg/L)。Using DMSO and amphotericin B as controls, the above compounds had no obvious inhibitory effect on Candida albicans (MIC value was ≥50 mg/L).
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。The above description of the embodiments is to facilitate those of ordinary skill in the technical field to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without inventive efforts. Therefore, the present invention is not limited to the above embodiments. Based on the disclosure of the present invention, improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.
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