Identification of the putative bryostatin polyketide synthase gene cluster from "Candidatus Endobugula sertula", the uncultivated microbial symbiont of the marine bryozoan Bugula neritina

The bryostatins are protein kinase C modulators with unique structural features and potential anticancer and neurological activities. These complex polyketides were isolated from the marine bryozoan Bugula neritina, but recent studies indicate that they are produced by the uncultured symbiotic bacterium "Candidatus Endobugula sertula" ("E. sertula"). Here we present the putative biosynthetic genes: five modular polyketide synthase (PKS) genes, a discrete acyltransferase, a beta-ketosynthase, a hydroxy-methyl-glutaryl CoA synthase (HMG-CS), and a methyltransferase. The cluster was sequenced in two closely related "E. sertula" strains from different host species. In one strain the gene cluster is contiguous, while in the other strain it is split into two loci, with one locus containing the PKS genes and the other containing the accessory genes. Here, we propose a hypothesis for the biosynthesis of the bryostatins. Thirteen PKS modules form the core macrolactone ring, and the pendent methyl ester groups are added by the HMG-CS gene cassette. The resulting hypothetical compound bryostatin 0 is the common basis for the 20 known bryostatins. As "E. sertula" is to date uncultured, heterologous expression of this biosynthetic gene cluster has the potential of producing the bioactive bryostatins in large enough quantities for development into a pharmaceutical.     

Alkaloids from amphibian skin: a tabulation of over eight-hundred compounds

A diverse array of biologically active, lipid-soluble alkaloids have been discovered in amphibian skin. Such alkaloids include the following: the steroidal samandarines from salamanders, the batrachotoxins, histrionicotoxins, gephyrotoxins, and epibatidine from neotropical poison frogs (Dendrobatidae), the pumiliotoxins, allopumiliotoxins, homopumiliotoxins, and decahydroquinolines from certain genera of anurans from four families (Dendrobatidae, Mantellidae, Bufonidae, and Myobatrachidae), a variety of izidines (pyrrolizidines, indolizidines, quinolizidines, lehmizidines), pyrrolidines, piperidines, various tricyclics (related in structures to the coccinellines), and spiropyrrolizidines from the first three of these four families, the pseudophrynamines from one genus of Australian frogs, and a variety of unclassified alkaloids as yet of undetermined structure. With the exception of the samandarines and the pseudophrynamines, all alkaloids appear to be derived from dietary sources. Although only a few of the over 800 amphibian skin alkaloids have been detected in arthropods, putative arthropod sources for the batrachotoxins and coccinelline-like tricyclics (beetles), the pumiliotoxins (ants, mites), the decahydroquinolines, izidines, pyrrolidines, and piperidines (ants), and the spiropyrrolizidines (millipedes) have been discovered. Ants are likely sources for histrionicotoxins, lehmizidines, and tricyclic gephyrotoxins. Epibatidines represent an important alkaloid class without a putative dietary source. The structures for many of these alkaloids have been rigorously established, while the structures of others represent tentative proposals, based only on mass spectral and FTIR spectral data, along with analogies to structures of well-defined alkaloids.     

川贝母转录组密码子使用偏好性分析

研究川贝母表达基因的密码子的使用模式,探讨其密码子使用偏好性特点,为运用基因工程技术实现贝母碱的异源生物合成提供理论依据。以川贝母转录组9 843条全长转录本序列为数据来源,使用Codon W软件对川贝母表达基因的密码子GC含量特点、有效密码子数和同义密码子相对使用频率各项参数进行计算、统计,分析发现,川贝母表达基因密码子偏好程度整体水平不高。该研究确定了15个密码子为川贝母最优密码子UUG,CUU,AUU,GUU,UCA,CCU,CCA,ACU,ACA,GCA,UAU,CAU,AAU,AGA和GGA,最优密码子偏好A/T结尾。计算结果显示26个川贝母生物碱合成相关基因中大肠杆菌和酿酒酵母稀有密码子所占比例较低。根据稀有密码子比例,逐个分析26个关键酶基因在大肠杆菌和酿酒酵母体系中表达时可采用的密码子优化方案。该研究为通过合成生物学手段异源合成川贝母生物碱奠定了前期研究基础。     

New alkaloids of the sarpagine group from Rauvolfia serpentina hairy root culture

Three new monoterpenoid indole alkaloids, 19(S),20(R)-dihydroperaksine (1), 19(S),20(R)-dihydroperaksine-17-al (2), and 10-hydroxy-19(S),20(R)-dihydroperaksine (3), along with 16 known alkaloids 4-19 were isolated from hairy root culture of Rauvolfia serpentina, and their structures were elucidated by 1D and 2D NMR analyses. Taking into account the stereochemistry of the new alkaloids and results of preliminary enzymatical studies, the putative biosynthetical relationships between the novel alkaloids are discussed.     

Clavicipitaceous fungi associated with ergoline alkaloid-containing convolvulaceae

Ergoline alkaloids are a group of physiologically active natural products occurring in taxonomically unrelated fungal and plant taxa Clavicipitaceae (Hypocreales) and Convolvulaceae (Solanales). We show in the present paper that clavicipitaceous fungi are associated with four different ergoline alkaloid-containing plant taxa of the family Convolvulaceae. These fungi are macroscopically visible on the adaxial surface when young leaf buds are opened or are detectable by molecular biological techniques in seeds. Detectability of the fungus correlates with the absence or presence of ergoline alkaloids within the respective plant organ. The fungi contain the gene (dmaW) responsible for the committed step in ergoline alkaloid biosynthesis. Sequencing of ribosomal DNA (18S rDNA and internal transcribed spacer) as well as the dmaW gene (partial) and construction of phylogenetic trees show that the fungi are clavicipitaceous, not identical but very closely related.     

卷叶贝母环阿屯醇合成酶基因的克隆及表达分析

目的对卷叶贝母Fritillaria cirrhosa中参与生物碱合成的关键酶环阿屯醇合成酶(cycloartenol synthase,CAS)基因进行克隆,并对其进行生物信息学和表达分析。方法基于转录组测序结果,通过PCR技术克隆卷叶贝母CAS基因(FcCAS)开放阅读框(open reading frame,ORF)序列,并基于在线工具对cDNA序列进行生物信息学分析。通过荧光定量(q RT-PCR)手段检测FcCAS在野生鳞茎与愈伤组织(通过激素组合刺激获得的组织培养物)中的表达情况并测定总生物碱的含量。结果 FcCAS编码区ORF长为2 271 bp,编码756个氨基酸,并与NCBI上公布的天门冬、芭蕉、油棕等植物CAS蛋白的相似性达80%以上;qRT-PCR与总生物碱含量测定实验表明FcCAS的表达水平与总生物碱含量的变化趋势一致,都是愈伤组织高于野生鳞茎。结论 FcCAS在不同组织状态下表达量差异较大,FcCAS是一个有生物学功能的蛋白质,受激素组合诱导表达,为进一步研究CAS对卷叶贝母生物碱含量的影响和表达调控奠定基础。     

长春花密码子使用偏好性分析

以长春花为研究对象,分析其密码子使用偏好性,以期为相关基因的异源表达、基因的预测、物种的进化研究提供指导。该研究以长春花的30 437条蛋白质编码序列为数据来源,对长春花密码子组成和密码子偏性的各项参数进行了计算和统计分析。计算了长春花萜类吲哚生物碱(terpenoid indole alkaloids,TIAs)生物合成途径中25个关键酶基因含有大肠杆菌或酿酒酵母稀有密码子的比例。结果显示,长春花基因的平均GC量为42.47%,密码子第3位碱基平均GC量为35.89%。长春花中共有28个密码子的同义密码子相对使用度(relative synonymous codon usage,RSCU)大于1,其中26个以A或T结尾。25个关键酶基因含有大肠杆菌稀有密码子的比例明显高于酿酒酵母稀有密码子的比例。长春花主要偏爱使用以A和T结尾的密码子;相比于酿酒酵母,其密码子使用特点与大肠杆菌的差异更大,推测酿酒酵母可能是长春花基因更合适的异源表达宿主。     

UGT88B2: A promiscuous O-glycosyltransferase from Carthamus tinctorius

Objective: In order to obtain new glycosyltransferases with highly efficient catalysis, the glycosyltransferases from Carthamus tinctorius which contains diverse types of glycosides were mined. Methods: A new glycosyltransferase gene (UGT88B2) with full length was obtained by PCR and further transformed into Escherichia coli for heterologous expression. The catalytic activity of recombinant UGT88B2 was determined by HPLC-MSn. The structures of representative catalytic products were elucidated by MS and NMR. Results: UGT88B2 exhibited catalytic promiscuity and various patterns in glycosylation of flavonoids with high efficiency. Conclusion: A new glycosyltransferase named UGT88B2 was successfully mined and can be employed as enzymatic tools in glycosylation of flavonoids.     

海绵Ircinia sp.中几种含溴生物碱的分离与鉴定

目的:研究海绵Ircinia sp.中生物碱成分。方法:通过多种色谱手段,对海绵Ircinia sp.中的生物碱成分进行分离纯化,根据波谱数据和理化性质确定化合物的结构。结果:从海绵Ircinia sp.中共分离得到5个含溴生物碱,分别鉴定为:fistularin-1(1)、fistularin-3(2)、aerothionin(3)、11-hydroxyaerothionin(4)、suberamolline A(5)。结论:以上5个生物碱均为首次从中国南海海绵Ircinia sp.中分离得到。     

苦豆子不同部位总生物碱提取物指纹图谱分析

目的:研究苦豆子不同部位总生物碱提取物的HPLC指纹图谱,为科学评价及有效控制苦豆子总生物碱提取物的质量提供依据。方法:采用HPLC-DAD方法,对不同主产区的16批苦豆子种子(苦豆籽)和地上部分(苦豆草)总生物碱提取物进行指纹图谱研究,采用相似度评价对指纹图谱进行分析,同时运用聚类分析及主成分分析对其进行模式识别。结果:分别建立苦豆籽和苦豆草提取物对照指纹图谱,确定共有峰,并指认色谱峰。通过聚类分析和主成分分析将16批药材总生物碱提取物按不同产地分为3类。结论:苦豆籽和苦豆草提取物的指纹图谱特征性及专属性良好,可用于全面控制苦豆籽和苦豆草总生物碱的质量。     

蝙蝠葛酚性碱对BxPC-3荷瘤小鼠细胞凋亡基因p53表达的影响

目的:观察蝙蝠葛酚性碱(PAMD)的抗肿瘤作用并进一步探讨其作用机制。方法:通过实时定量PCR(Real-time PCR)技术观察PAMD对胰腺癌细胞株BxPC-3皮下异位移植瘤细胞凋亡相关基因p53mRNA表达的影响,以此探讨PAMD抗肿瘤作用机理。结果:PAMD能够显著降低胰腺癌细胞株BxPC-3裸鼠皮下异位移植瘤组织中p53 mRNA表达量。结论:PAMD对胰腺癌细胞株BxPC-3裸鼠皮下异位移植瘤表现出明显抑制作用,能够有效的抑制肿瘤生长,其机理可能与凋亡相关基因表达有关。     

乌梅中生物碱的分离与鉴定

乌梅经80%乙醇浸提后用氯仿萃取浓缩,转入水相通过大孔阳离子交换树脂柱,洗脱液用氯仿萃取浓缩后,石油醚溶解进行重结晶,升华提纯,IR、MS、1HNMR、13CNMR鉴定化合物的结构.从中分离并鉴定了2个生物碱为:2,2,6,6-四甲基哌啶酮(Ⅰ),叔丁基脲(Ⅱ).化合物Ⅰ、Ⅱ均为乌梅中新发现的生物碱.     

Two Alkaloids from Bulbs of Lycoris sanguinea MAXIM. Suppress PEPCK Expression by Inhibiting the Phosphorylation of CREB

In the fasting state, gluconeogenesis is upregulated by glucagon. Glucagon stimulates cyclic adenosine monophosphate production, which induces the expression of key enzymes for gluconeogenesis, such as cytosolic phosphoenolpyruvate carboxykinase (PEPCK‐C), which are involved in gluconeogenesis through the protein kinase A/cAMP response element‐binding protein (CREB) pathway. Using a luciferase reporter gene assay, a methanol extract of the bulbs of Lycoris sanguinea M_AXIM. var. kiushiana Makino was found to suppress cAMP‐enhanced PEPCK‐C promoter activity. In addition, two alkaloids, lycoricidine and lycoricidinol, in the extract were identified as active constituents. In forskolin‐stimulated human hepatoma cells, these alkaloids suppressed the expression of a reporter gene under the control of cAMP response element and also prevented increases in the endogenous levels of phosphorylated CREB and PEPCK mRNA expression. These results suggest that lycoricidine and lycoricidinol suppress PEPCK‐C expression by inhibiting the phosphorylation of CREB and may thus have the potential to prevent excessive gluconeogenesis in type 2 diabetes. Copyright © 2016 John Wiley & Sons, Ltd.     

Cloning and biochemical characterization of the hectochlorin biosynthetic gene cluster from the marine cyanobacterium Lyngbya majuscula

Cyanobacteria, or blue-green algae, are a rich source of novel bioactive secondary metabolites that have potential applications as antimicrobial or anticancer agents or useful probes in cell biology studies. A Jamaican collection of the cyanobacterium Lyngbya majuscula has yielded several unique compounds including hectochlorin ( 1) and the jamaicamides A-C ( 5- 7). Hectochlorin has remarkable antifungal and cytotoxic properties. In this study, we have isolated the hectochlorin biosynthetic gene cluster ( hct) from L. majuscula to obtain details regarding its biosynthesis at the molecular genetic level. The genetic architecture and domain organization appear to be colinear with respect to its biosynthesis and consists of eight open reading frames (ORFs) spanning 38 kb. An unusual feature of the cluster is the presence of ketoreductase (KR) domains in two peptide synthetase modules, which are predicted to be involved in the formation of the two 2,3-dihydroxyisovaleric acid (DHIV) units. This biosynthetic motif has only recently been described in cereulide, valinomycin, and cryptophycin biosynthesis, and hence, this is only the second such report of an embedded ketoreductase in a cyanobacterial secondary metabolite gene cluster. Also present at the downstream end of the cluster are two cytochrome P450 monooxygenases, which are likely involved in the formation of the DHIV units. A putative halogenase, at the beginning of the gene cluster, is predicted to form 5,5-dichlorohexanoic acid.     

钩枝藤总生物碱对非小细胞肺癌细胞系表达谱及免疫微环境的影响

目的 研究钩枝藤总生物碱对非小细胞肺癌细胞系表达谱的影响,筛选出差异表达基因（differentially expressed genes,DEGs）并对功能基因进行分析和挖掘,以期为钩枝藤抗肺癌的分子机制提供理论依据。方法 将非小细胞肺癌NCI-H1975细胞分为对照组和钩枝藤总生物碱处理组,使用RNA-Seq技术开展全转录组测序,获取DEGs;对DEGs进行基因本体（gene ontology,GO）功能及京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes,KEGG）通路富集分析;筛选差异基因核心模块并分析其影响功能,进一步分析关键基因对非小细胞肺癌生存期的影响;通过分析钩枝藤总生物碱对非小细胞肺癌免疫细胞肿瘤浸润的情况,确定关键基因表达与免疫浸润水平的相关性。结果 对照组和药物组共检测到2976个DEGs,其中药物组664个DEGs上调,2312个DEGs下调。钩枝藤总生物碱对铁死亡、氧化磷酸化、线粒体、溶酶体、细胞间隙连接、核苷酸结合寡聚化结构域（nucleotide binding oligomerization domain,NOD）样受体等介导的信号通路产生重要影响。在DEGs核心模块中,白细胞介素11（interleukin 11,IL11）的高表达可显著影响非小细胞肺癌患者的生存期,是非小细胞肺癌患者不良预后的关键因素之一;角形四肽重复干扰素诱导蛋白3（interferon induced protein with tetratricopeptide repeats 3,IFIT3）、白细胞介素7受体（interleukin 7 receptor,IL7R）、2'',5''-寡腺苷酸合成酶2（2'',5''-oligoadenylate synthetase 2,OAS2）、S-腺苷甲硫氨酸基结构域蛋白2（radicalS-adenosyl methionine domain containing 2,RSAD2）、受体转运蛋白4（receptor transporter protein 4,RTP4）、不育α基序结构域9样（sterile alpha motif domain containing 9-like gene,SAMD9L）、X染色体连锁凋亡抑制蛋白相关因子1（X-linked inhibitor of apoptosis associated factor 1,XAF1）的表达水平影响了非小细胞肺癌组织的纯度和免疫细胞浸润的类型,可能改变非小细胞肺癌的免疫微环境。结论 钩枝藤总生物碱能够影响非小细胞肺癌细胞的转录谱,具有潜在的抗非小细胞肺癌活性作用。     

Study on the Biological Basis of Hypertension and Syndrome with Liver-Fire Hyperactivity Based on Data Mining Technology

Objective: To construct gene co-occurrence network of hypertension and liver-fire hyperactivity syndrome, to investigate the biological basis of hypertension and liver-fire hyperactivity syndrome and the characteristics of the molecular network from gene level. Materials and Methods: Applying GenCLip 2.0 online platform to retrieve the up-to-date literature referred to essential hypertension from PubMed database, cluster the abnormal expression of essential hypertension-related genes and analyze their function, combining Kyoto encyclopedia of genes and genomes-pathway analysis to investigate the closely related genes and the signaling molecules. Based on the genes closely related to hypertension, standard diagnostic symptoms of liver?fire hyperactivity were used as keywords to conduct hypertension liver?fire hyperactivity?related gene cluster analysis. Results: The top 1000 genes of essential hypertension were retrieved from GenCLip 2.0 online platform, which mainly clustered in the regulation of ambulatory blood pressure, regulation of renin-angiotensin-aldosterone system (RAAS), and sympathetic nervous system activity, as well as endothelial dysfunction; the closely related genes of hypertension with liver?fire hyperactivity are related to RAAS, gene REN, angiotensin converting enzyme, angiotensinogen, and cytochrome P450 family CYP2D6. Conclusion: A combination of literature mining and data mining can construct the gene network of hypertension and the syndrome-related genes, which provides a new method for the study of the biological basis of hypertension from the genetic level.     

Biosynthetic pathway and gene cluster analysis of curacin A, an antitubulin natural product from the tropical marine cyanobacterium Lyngbya majuscula

Curacin A (1) is a potent cancer cell toxin obtained from strains of the tropical marine cyanobacterium Lyngbya majuscula found in Cura?ao. Its structure is unique in that it contains the sequential positioning of a thiazoline and cyclopropyl ring, and it exerts its potent cell toxicity through interaction with the colchicine drug binding site on microtubules. A series of stable isotope-labeled precursors were fed to cultures of curacin A-producing strains and, following NMR analysis, allowed determination of the metabolic origin of all atoms in the natural product (one cysteine, 10 acetate units, two S-adenosyl methionine-derived methyl groups) as well as several unique mechanistic insights. Moreover, these incorporation experiments facilitated an effective gene cloning strategy that allowed identification and sequencing of the approximately 64 kb putative curacin A gene cluster. The metabolic system is comprised of a nonribosomal peptide synthetase (NRPS) and multiple polyketide synthases (PKSs) and shows a very high level of collinearity between genes in the cluster and the predicted biochemical steps required for curacin biosynthesis. Unique features of the cluster include (1) all but one of the PKSs are monomodular multifunctional proteins, (2) a unique gene cassette that contains an HMG-CoA synthase likely responsible for formation of the cyclopropyl ring, and (3) a terminating motif that is predicted to function in both product release and terminal dehydrative decarboxylation.     

中国市场柏子仁真菌污染调查研究

中药柏子仁在采收、生产加工、储藏、运输等环节中,受自身及环境因素的影响极易发生霉变,滋生产毒真菌或致病真菌,甚至产生真菌毒素,影响临床用药安全。2020年版《中国药典》限制柏子仁中黄曲霉毒素含量最高标准。然而,对于柏子仁药材上的真菌污染状况鲜有研究,难以有针对性地进行防控。因此,该文基于Illumina NovaSeq6000平台,利用ITS序列扩增子技术分析了我国市场上27批市售柏子仁的真菌分布及多样性。从中共鉴定出10门,35纲,93目,193科,336属,372种真菌。其中曲霉属、链格孢属占主要优势,有20批样本检测出黄曲霉菌,10批样本检测出构巢曲霉菌,且所有样本均检测出烟曲霉和黑曲霉等潜在致病真菌。经多样性分析,甘肃省所采样本真菌群落多样性程度高,山东省所采样本所含真菌物种数量最多,广西所采样本表面真菌群落多样性最低且物种数最少。大部分样品中检测出了不同程度的烟曲霉、黑曲霉、黄曲霉、寄生曲霉等致病真菌。该研究从柏子仁产业链的最后环节市场上系统调查了我国柏子仁真菌污染状况,明确了柏子仁真菌尤其是产毒真菌分布,为柏子仁中真菌污染的靶向防控提供理论依据。     

昆明山海棠总生物碱诱导小鼠淋巴瘤细胞tk基因突变的研究

目的 :研究昆明山海棠 (THH)总生物碱对小鼠淋巴瘤L5 178Y细胞tk基因的影响。方法 :用抽提的THH总生物碱对L5 178Y细胞进行浓度梯度染毒 ,不同时间点进行单细胞微孔接种 ,计数阳性集落 ,测定细胞接种效率、相对存活率、相对悬浮增长率和突变频率。结果 :随着THH总生物碱作用剂量的增加 ,L5 178Y细胞相对存活率和相对悬浮增长率均明显下降 ;在 0.1～2 .0 g·L^-1范围内可诱导细胞tk位点的突变 ,诱发突变频率是自发突变频率的 2～9倍。在tk位点诱发了两种不同表型的突变集落 ,即大集落与小集落 ,但以大集落为主 ,这一现象可能与THH的作用机制有关。结论 :THH总生物碱对L5 178Y细胞具有肯定的细胞毒性与诱导基因突变作用。