A new acyclic thiophene sesterterpene from the Sikao Bay sponge,Xestospongia sp.

Bioactivity-guided fractionation of the ethyl acetate extract of a marine sponge, Xestospongia sp., led to the isolation of a new thiophene-S-oxide acyclic sesterterpene (1). The chemical structure was extensively analyzed using NMR and mass spectral data. Compound 1 showed weak cytotoxicity against Vero cells.     

Systematic mining of fungal chimeric terpene synthases using an efficient precursor-providing yeast chassis

Chimeric terpene synthases, which consist of C-terminal prenyltransferase (PT) and N-terminal class I terpene synthase (TS) domains (termed PTTSs here), is unique to fungi and produces structurally diverse di- and sesterterpenes. Prior to this study, 20 PTTSs had been functionally characterized. Our understanding of the origin and functional evolution of PTTS genes is limited. Our systematic search of sequenced fungal genomes among diverse taxa revealed that PTTS genes were restricted to Dikarya. Phylogenetic findings indicated different potential models of the origin and evolution of PTTS genes. One was that PTTS genes originated in the common Dikarya ancestor and then underwent frequent gene loss among various subsequent lineages. To understand their functional evolution, we selected 74 PTTS genes for biochemical characterization in an efficient precursor-providing yeast system employing chassis-based, robot-assisted, high-throughput automatic assembly. We found 34 PTTS genes that encoded active enzymes and collectively produced 24 di- and sesterterpenes. About half of these di- and sesterterpenes were also the products of the 20 known PTTSs, indicating functional conservation, whereas the PTTS products included the previously unknown sesterterpenes, sesterevisene (1), and sesterorbiculene (2), suggesting that a diversity of PTTS products awaits discovery. Separating functional PTTSs into two monophyletic groups implied that an early gene duplication event occurred during the evolution of the PTTS family followed by functional divergence with the characteristics of distinct cyclization mechanisms.

Terpenoids comprise the largest and most structurally diverse family of natural products (>80,000 have been characterized), and they are produced by organisms in all domains of life (1, 2). These organic chemicals play various roles in defense, regulation, and communication and have served as therapeutics, fragrances, and flavorings (3, 4). Terpenoids are biosynthesized from the universal C5 precursors, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), which are produced via the mevalonate and/or methyl-erythritol 4-phosphate pathways (depending on the organism). Subsequently, prenyltransferases (PTs) assemble IPP and DMAPP into linear isoprenyl diphosphate chains of various lengths. Terpene synthases (TSs) use these chains to generate a hydrocarbon core skeleton with multiple chiral centers. The core skeleton is modified by a series of enzymes to generate structurally and functionally diverse terpenoids (SI Appendix, Fig. S1). The structural complexity and corresponding diversity of terpenoids is primarily due to various TSs. According to the initial carbocation formation strategy, TSs are traditionally classified into a class I TS clade that generates carbocation via diphosphate ionization and a class II clade that initiates cyclization by protonating an olefinic double bond or epoxide group in an isoprenoid substrate (5–7). Class I TSs have the characteristic conserved metal-binding, Asp-rich structural motifs, DDXXD/E and (N/H)DXX(S/T)XXXE, whereas class II TSs have the signature of a general acid motif, DXDD (8, 9). PTs are generally divided into cis-PTs and trans-PTs, with trans-PTs containing two aspartate-rich motifs (10). Despite the absence of significant sequence similarity, trans-PTs and class I TSs are evolutionarily related (11), sharing a conserved structural domain known as the TS fold or alpha domain (9).Some enzymes are inherently chimeric, and the close physical proximity of their active sites can enhance product metabolic flux (12, 13). Chimeric TSs comprising a C-terminal PT domain and a class I N-terminal TS domain (PTTSs) represent a rare and important class of TSs found in various species of fungi. These PTTSs can directly use IPP and DMAPP as cosubstrates to form diverse di- and sesterterpene skeletons (14). Since the discovery of the chimeric diterpene synthase PaFS (15) and the chimeric sesterterpene synthase AcOS (16), ∼20 PTTSs and their resulting di- and sesterterpenes have been characterized using genome-based approaches (SI Appendix, Table S1). Notably, most compounds produced by PTTSs have novel basic carbon skeletons and complex biosynthetic mechanisms, including 5/6/3/6/5 pentacyclic preasperterpenoid A (17), 5/8/6/5 tetracyclic sesterfisherol (18), 5/5/5/6/5 pentacyclic quiannulatene (19), and 5/6/5/5 tetracyclic phomopsene (20) (Fig. 1). According to the initial carbocation formation strategy, the cyclization mechanisms of PTTSs can be classified into a type A (C1-IV-V) mode in which sequential cyclization is initiated between the C1-C15/C14-C18 of geranylfarnesyl diphosphate (GFPP) to yield a five to 15 ring system and a type B (C1-III-IV) mode in which a five to 11 ring system is uniformly generated by cyclization at the C1-C11/C10-C14 of GFPP/geranylgeranyl diphosphate (GGPP) (14, 21).Open in a separate windowFig. 1.Chemical structures of di- and sesterterpenes synthesized by known fungal PTTSs and the general PTTS profile. The origin and corresponding PTTSs for di- and sesterterpenes are shown. For PTTS, the prenyltransferase domain can catalyze the condensation of IPP and DMAPP to produce GGPP and GFPP. Then, the TS domain utilizes GGPP or GFPP as a substrate to generate cyclic and acyclic di- and sesterterpenes.The accumulating number of PTTSs requiring functional characterization has generated several fundamental questions that await answers, such as whether PTTS genes of interest are widely distributed among fungi or restricted to specific groups and which mechanisms govern their functional evolution. Numerous PTTSs need to be characterized to answer these questions, and this requires an efficient high-throughput system, although Aspergillus oryzae is widely used for heterologous expression and it has facilitated the characterization of a series of PTTSs from filamentous fungi (18, 22). However, rapidly characterizing the functions of numerous unknown PTTSs would be impossible using this system. The development of robust terpene precursor-providing chassis in Escherichia coli and Saccharomyces cerevisiae has provided efficient approaches for the overproduction of farnesene, taxadiene, and artemisinic acid (23–25) and a scalable strategy for rapidly characterizing the functions of TSs, accelerating the process of mining novel sesqui-, di-, and sesterterpenes with unusual skeletons (26–29).In this study, we systematically collected PTTS genes from various fungal species and propose an evolutionary model in which PTTS genes have a common origin in the Dikarya ancestor. Gene duplication and frequent gene loss are among the inferred mechanisms governing PTTS gene evolution. We then determined the catalytic functions of 34 PTTSs using a robot-based automatic high-throughput assembly platform and an efficient precursor-providing S. cerevisiae chassis. Thus, our findings provide profound insights into the origin and functional evolution of PTTSs, a class of TS genes that are unique to fungi.     

西沙海绵Dysidea granulosa 化学成分研究

目的 研究中国西沙群岛海绵Dysidea granulosa的化学成分。 方法 利用薄层色谱、硅胶色谱、Sephadex LH-20色谱、MPLC、半制备HPLC等方法对化学成分进行分离纯化;通过NMR、MS等方法,并结合文献,鉴定化合物的结构。结果 从西沙海绵Dysidea granulosa的甲醇粗提取物中,分离得到6个化合物： Honulactone A (1), Honulactone B(2), Honulactone E (3), Honulactone F (4), 2-(2’,4’-Dibromophenoxy)-4,6-dibromophenol (5), 3,5-Dibromo-6-chloro-2-(2,4- dibromophenoxy)phenol (6)。结论 化合物1、2、3、4均为首次从该种海绵中分离得到。