珊瑚来源真菌Parengyodontium album SCSIO SX7W11中聚酮化合物的发现及其生物合成途径分析

海洋来源真菌的天然产物因其独特的结构与生物学活性而备受关注,而利用基因组信息对其代谢产物进行深入挖掘也成为研究策略之一。[目的] 本文以一株南海珊瑚来源的真菌Parengyodontium album SCSIO SX7W11为目标菌株,挖掘其生产聚酮类化合物的潜能。[方法] 本研究利用Illumina Miseq技术对SX7W11菌株进行全基因组扫描测序,运用生物信息学手段对其基因组的生物合成基因簇进行预测和基因功能注释,挖掘可能产生新颖聚酮化合物的基因簇。对SX7W11进行放大发酵后,利用正相色谱、中压反相色谱、Sephadex LH-20凝胶色谱、HPLC半制备等分离手段分离纯化出单体化合物。再利用高分辨质谱（HR-ESI-MS）、¹H NMR、¹³C NMR、X-ray单晶衍射等波谱手段确定化合物的结构,并根据生物合成基因簇对化合物的生物合成途径进行推导。[结果] 全基因组扫描测序结果显示,P.album SCSIO SX7W11基因组大小为34.0 Mb,含有24个生物合成基因簇,包括6个聚酮合酶基因簇以及3个萜烯合酶基因簇。从发酵产物中分离鉴定到3个聚酮类化合物：emodin（1）、alternaphenol B（2）和sydowinin A（3）,其中化合物3获得了单晶结构数据。通过生物信息学方法从菌株基因组中定位到了sydowinin A的生物合成基因簇。结合文献对emodin（1）、alternaphenol B（2）和sydowinin A（3）的生物合成途径进行了分析。[结论] 本研究通过基因组挖掘及培养基优化,发现1株珊瑚来源的真菌P.album SCSIO SX7W11具有生产sydowinins类聚酮类化合物的能力,为该类化合物生物合成机制深入研究奠定了基础。     

Underestimated biodiversity as a major explanation for the perceived rich secondary metabolite capacity of the cyanobacterial genus Lyngbya

Marine cyanobacteria are prolific producers of bioactive secondary metabolites responsible for harmful algal blooms as well as rich sources of promising biomedical lead compounds. The current study focused on obtaining a clearer understanding of the remarkable chemical richness of the cyanobacterial genus Lyngbya. Specimens of Lyngbya from various environmental habitats around Curaçao were analysed for their capacity to produce secondary metabolites by genetic screening of their biosynthetic pathways. The presence of biosynthetic pathways was compared with the production of corresponding metabolites by LC‐ESI‐MS² and MALDI‐TOF‐MS. The comparison of biosynthetic capacity and actual metabolite production revealed no evidence of genetic silencing in response to environmental conditions. On a cellular level, the metabolic origin of the detected metabolites was pinpointed to the cyanobacteria, rather than the sheath‐associated heterotrophic bacteria, by MALDI‐TOF‐MS and multiple displacement amplification of single cells. Finally, the traditional morphology‐based taxonomic identifications of these Lyngbya populations were combined with their phylogenetic relationships. As a result, polyphyly of morphologically similar cyanobacteria was identified as the major explanation for the perceived chemical richness of the genus Lyngbya, a result which further underscores the need to revise the taxonomy of this group of biomedically important cyanobacteria.     

藤黄生孢链霉菌NRRL 2401遗传操作系统和基因文库的构建北大核心CSCD

【目的】建立藤黄生孢链霉菌NRRL 2401的遗传操作系统和基因文库,以便筛选次级代谢产物生物合成基因。【方法】利用大肠杆菌和链霉菌的属间接合转移的方法,以整合型载体pPM927、pSET152和复制型载体pJTU1278构建链霉菌遗传操作系统。以pCClFOS^(TM)载体,大肠杆菌EP1300^(TM)-T1~R为宿主菌构建Fosmid文库。随后,设计引物,利用"板池-行池-单克隆"的三级PCR方法对文库进行快速筛选。【结果】pPM927、pSET152和pJTU1278均成功转入藤黄生孢链霉菌NRRL 2401,其中pSET152载体的转化效率最高。构建了稳定高效的藤黄生孢链霉菌NRRL 2401的基因文库,含2880个克隆,平均插入片段大小约为35 kb,空载率小于1%,文库覆盖率为99.99%,覆盖基因组16.5倍。同时,初步筛选出可能含有吲哚霉素生物合成基因簇的9个阳性克隆。【结论】成功构建了稳定高效的藤黄生孢链霉菌NRRL 2401遗传操作系统和高质量的基因文库,为克隆该菌中次级代谢产物生物合成基因簇以及进一步遗传改造奠定了基础。     

A radiochemical technique with potential for revealing novel fungal metabolites according to expression of specific biosynthetic activities

Fungal secondary metabolites are mostly derived from a few key intermediates in primary metabolism, such as acetate and some amino acids. Classical screens for novel fungal compounds of possible industrial interest have used chromatographic displays of extract components, as was the practice for plant natural products, followed by structural determination and pharmacological study. In contrast, modern robotic screens usually focus initially on specific bioassay applied to fermentation products and crude extracts. Both strategies are expensive in terms of human resources and/or in sophisticated equipment. A relatively inexpensive technique, exploiting biosynthetic principles through use of ¹⁴C-labelled probes to recognise particular structural features by autoradiography of tlc plates is described and discussed. Application to 80 isolates of filamentous fungi from Caribbean and European/Mediterranean environments enabled recognition of arrays of metabolites according to their biosynthetic origin, showing the potential of the technique in novel product discovery in unsophisticated laboratory facilities, as exemplified by reference to subsequent discovery of novel metabolites produced by Amorosia littoralis.     

Biochemical synthesis of uniformly 13C-labeled diterpene hydrocarbons and their bioconversion to diterpenoid phytoalexins in planta

Phytocassanes and momilactones are the major diterpenoid phytoalexins inductively produced in rice as bioactive substances. Regardless of extensive studies on the biosynthetic pathways of these phytoalexins, bioconversion of diterpene hydrocarbons is not shown in planta. To elucidate the entire biosynthetic pathways of these phytoalexins, uniformly ¹³C-labeled ent-cassadiene and syn-pimaradiene were enzymatically synthesized with structural verification by GC–MS and ¹³C-NMR. Application of the ¹³C-labeled substrates on rice leaves led to the detection of ¹³C-labeled metabolites using LC-MS/MS. Further application of this method in the moss Hypnum plumaeforme and the nearest out-group of Oryza species Leersia perrieri, respectively, resulted in successful bioconversion of these labeled substrates into phytoalexins in these plants. These results demonstrate that genuine biosynthetic pathways from these diterpene hydrocarbons to the end product phytoalexins occur in these plants and that enzymatically synthesized [U-¹³C₂₀] diterpene substrates are a powerful tool for chasing endogenous metabolites without dilution with naturally abundant unlabeled compounds.     

Gene expression in the scleractinian Acropora microphthalma exposed to high solar irradiance reveals elements of photoprotection and coral bleaching

Background

The success of tropical reef-building corals depends on the metabolic co-operation between the animal host and the photosynthetic performance of endosymbiotic algae residing within its cells. To examine the molecular response of the coral Acropora microphthalma to high levels of solar irradiance, a cDNA library was constructed by PCR-based suppression subtractive hybridisation (PCR-SSH) from mRNA obtained by transplantation of a colony from a depth of 12.7 m to near-surface solar irradiance, during which the coral became noticeably paler from loss of endosymbionts in sun-exposed tissues.

Methodology/Principal Findings

A novel approach to sequence annotation of the cDNA library gave genetic evidence for a hypothetical biosynthetic pathway branching from the shikimic acid pathway that leads to the formation of 4-deoxygadusol. This metabolite is a potent antioxidant and expected precursor of the UV-protective mycosporine-like amino acids (MAAs), which serve as sunscreens in coral phototrophic symbiosis. Empirical PCR based evidence further upholds the contention that the biosynthesis of these MAA sunscreens is a ‘shared metabolic adaptation’ between the symbiotic partners. Additionally, gene expression induced by enhanced solar irradiance reveals a cellular mechanism of light-induced coral bleaching that invokes a Ca²⁺-binding synaptotagmin-like regulator of SNARE protein assembly of phagosomal exocytosis, whereby algal partners are lost from the symbiosis.

Conclusions/Significance

Bioinformatics analyses of DNA sequences obtained by differential gene expression of a coral exposed to high solar irradiance has revealed the identification of putative genes encoding key steps of the MAA biosynthetic pathway. Revealed also by this treatment are genes that implicate exocytosis as a cellular process contributing to a breakdown in the metabolically essential partnership between the coral host and endosymbiotic algae, which manifests as coral bleaching.     

Heterologous expression system in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> for fungal biosynthetic gene clusters of secondary metabolites

Fungal secondary metabolites have been considered promising resources in the search for novel bioactive compounds. Given the high potential of fungi as genetic resources, it is essential to find an efficient way to link biosynthetic genes to the product in a heterologous system, because many genes for the secondary metabolite in the original strain are silent under standard laboratory conditions. In a previous study, we constructed a heterologous expression system for a biosynthetic gene cluster using Aspergillus oryzae as the host. To make the host more versatile for the expression of secondary metabolism genes, the expression levels of a global regulator, laeA, were increased by placing the A. oryzae laeA gene under the control of the constitutive active pgk promoter. In the A. oryzae overexpressing laeA, two clusters of heterologous biosynthetic genes [the monacolin K (MK) gene cluster from Monascus pilosus and the terrequinone A (TQ) gene cluster from Aspergillus nidulans] were successfully overexpressed, resulting in the production of the corresponding metabolite, MK or TQ. The successful production of secondary metabolites belonging to different structural groups, namely MK as a polyketide and TQ as a hybrid of amino acid and isoprenoid, indicated that the laeA-enriched A. oryzae was a versatile host for the heterologous expression of the biosynthetic gene cluster.     

Coral—the world's most diverse symbiotic ecosystem

Zooxanthellate corals (i.e. those harbouring Symbiodinium) are the main builders of the world's shallow‐water marine coral reefs. They represent intimate diverse symbioses between coral animals, single‐celled photosynthetic dinoflagellates (Symbiodinium spp.), other microscopic eukaryotes, prokaryotes and viruses. Crabs and other crustaceans, worms, sponges, bivalves and hydrozoans, fishes, sea urchins, octopuses and sea stars are itinerant members of these ‘rainforests of the sea’. This review focuses on the biodiversity of scleractinian coral animals and their best studied microscopic epi‐ and endosymbionts. In relation to coral‐associated species diversity, Symbiodinium internal transcribed spacer region sequence types tally 10²–10³ or up to ~15 different operational taxonomic units (OTUs, or putative species at the 97% sequence identity level; this cut‐off was chosen based on intragenomic sequence diversity observed in monoclonal cultures) and prokaryotes (mostly bacterial) total 10²–10⁴ OTUs. We analysed all publically accessible 16S rRNA gene sequence data and found Gammaproteobacteria were extremely abundant, followed by Alphaproteobacteria. Notably, Archaea were poorly represented and ‘unassigned OTUs’ were abundant in data generated by high‐throughput DNA sequencing studies of corals. We outline and compare model systems that could be used in future studies of the coral holobiont. In our future directions, we recommend a global coral sampling effort including substantial attention being paid to method of coral tissue acquisition, which compartments (mucus, tissue, skeleton) to explore, broadening the holobiont members considered and linking biodiversity with functional investigations.     

An in vitro system to study cyclopeptide heterophyllin B biosynthesis in the medicinal plant <Emphasis Type="Italic">Pseudostellaria heterophylla</Emphasis>

Plant cyclopeptides are a large group of small molecule metabolites found in a wide variety of plants, including traditional Chinese medicinal plants. Many of the cyclopeptides have highly unusual structures and potent biological activities. However, the majority of the cyclopeptides have not been studied for their biosynthetic mechanisms. In this study, we have established a culture system for the biosynthetic study of heterophyllin B (HB), a cyclopeptide produced by the medicinal plant Pseudostellaria heterophylla. We first developed a shoot culture of P. heterophylla that produced HB consistently under laboratory conditions. Using ¹⁴C-labeled proline as tracer, we showed that labeled HB was produced by the cultured shoots, indicating that this system has de novo biosynthetic activity. Next, we chemically synthesized HB’s linear peptide precursor (LHB) and the N-acetyl cysteamine thioester of LHB (LHB-SNAC). When LHB-SNAC was incubated with total cell free extracts of the cultured shoots, a small amount of cyclized product (HB), in addition to the hydrolyzed product (LHB), was produced. The in vivo and in vitro results demonstrate the presence of an HB biosynthetic system, which provides insight into the molecular mechanism for plant cyclopeptide biosynthesis.     

Pseudopterosin biosynthesis—pathway elucidation,enzymology, and a proposed production method for anti-inflammatory metabolites from<Emphasis Type="Italic"> Pseudopterogorgia elisabethae</Emphasis>

The pseudopterosins are a family of diterpene pentosides isolated from the marine octocoral, Pseudopterogorgia elisabethae. These compounds possess non-steroidal anti-inflammatory and analgesic properties which have been shown to be greater than the industry standard, indomethacin. In our investigations, we are interested in examining the biosynthesis and enzymology of these compounds for the development of a biotechnological production method. We have isolated the pseudopterosin diterpene cyclase product, elisabethatriene, using a radioactivity-guided isolation. This has provided us with an assay to isolate the diterpene cyclase enzyme. The amino acid sequence of the purified diterpene cyclase will facilitate cloning and expression of the gene in a suitable host. In addition, we have identified over 25 novel diterpenes from one of our collections of P. elisabethae. Several of these compounds appear to be involved in pseudopterosin biosynthesis and are presently being evaluated as potential intermediates. These compounds have also been evaluated for anti-inflammatory activity and some possess greater activity than that of the pseudopterosins. We therefore propose a production method utilizing a combination of recombinant enzyme technology and synthetic methods/biocatalysis in order to produce one or more anti-inflammatory metabolites in P. elisabethae.     

Coral-crab association: a compact domain of a multilevel trophic system

Colonies of the Red Sea branching coral Stylophora pistillata were incubated in situ with radioactive carbon and Trapezia cymodoce crabs were introduced to the colonies, for one month each, up to 7 months after coral labelling. Zooxanthellar photosynthetic products were translocated to the crabs via host coral tissue. Based upon crab/coral tissue conversion from 53 crab samples, crabs possessed radioactive material equal to that recorded in 320–770 mm² of coral tissue (up to 2257 mm²). This material was translocated mainly by direct grazing on coral tissue rather than mucus collection. Ovigerous female crabs (39 specimens) accumulated significantly more labelled carbon than males, and up to 53 % of their radioactivity was concentrated in their reproductive organs. A pair of crabs dwelling in a coral colony consumed ca 130 cm² of host tissue per month (40–45 cm length of coral branches). This system represents a compact, obligatory multilevel trophic domain which also radiates horizontally by allocation of energy derived from algal photosynthesis into planula and zooea larva production, permitting the development of long trophic chains and a complex food web.     

Metabolic diversification of nitrogen‐containing metabolites by the expression of a heterologous lysine decarboxylase gene in Arabidopsis

Lysine decarboxylase converts l ‐lysine to cadaverine as a branching point for the biosynthesis of plant Lys‐derived alkaloids. Although cadaverine contributes towards the biosynthesis of Lys‐derived alkaloids, its catabolism, including metabolic intermediates and the enzymes involved, is not known. Here, we generated transgenic Arabidopsis lines by expressing an exogenous lysine/ornithine decarboxylase gene from Lupinus angustifolius (La‐L/ODC) and identified cadaverine‐derived metabolites as the products of the emerged biosynthetic pathway. Through untargeted metabolic profiling, we observed the upregulation of polyamine metabolism, phenylpropanoid biosynthesis and the biosynthesis of several Lys‐derived alkaloids in the transgenic lines. Moreover, we found several cadaverine‐derived metabolites specifically detected in the transgenic lines compared with the non‐transformed control. Among these, three specific metabolites were identified and confirmed as 5‐aminopentanal, 5‐aminopentanoate and δ‐valerolactam. Cadaverine catabolism in a representative transgenic line (DC29) was traced by feeding stable isotope‐labeled [α‐¹⁵N]‐ or [ε‐¹⁵N]‐l ‐lysine. Our results show similar ¹⁵N incorporation ratios from both isotopomers for the specific metabolite features identified, indicating that these metabolites were synthesized via the symmetric structure of cadaverine. We propose biosynthetic pathways for the metabolites on the basis of metabolite chemistry and enzymes known or identified through catalyzing specific biochemical reactions in this study. Our study shows that this pool of enzymes with promiscuous activities is the driving force for metabolite diversification in plants. Thus, this study not only provides valuable information for understanding the catabolic mechanism of cadaverine but also demonstrates that cadaverine accumulation is one of the factors to expand plant chemodiversity, which may lead to the emergence of Lys‐derived alkaloid biosynthesis.     

Predation of the sea urchin Diadema antillarum Philippi on living coral

Summary On the coral reefs of Curaçao and Bonaire (Netherlands Antilles) the sea urchin Diadema antillarum is a major coral predator. In areas with high coral cover, up to 8.2% of the Diadema population (with a density of 8.5 animals/m²) was feeding on living coral surfaces at night. Acropora species are the most heavily attacked corals.     

Identifying metabolic alterations associated with coral growth anomalies using 1H NMR metabolomics

Coral growth anomalies (GAs) are tumor-like protrusions that are detrimental to coral health, affecting both the coral skeleton and soft tissues. These lesions are increasingly found throughout the tropics and are commonly associated with high human population density, yet little is known about the molecular pathology of the disease. Here, we investigate the metabolic impacts of GAs through ¹H nuclear magnetic resonance (NMR) metabolomics in Porites compressa tissues from a site of high disease prevalence (Coconut Island, Hawaii). We putatively identified 18 metabolites (8.1% of total annotated features) through complementary ¹H and ¹H–¹³C heteronuclear single quantum correlation NMR data that increase confidence in pathway analyses and may bolster future coral metabolite annotation efforts. Extract yield was elevated in both GA and unaffected (normal tissue from a diseased colony) compared to reference (normal tissue from GA-free colony) samples, potentially indicating elevated metabolic activity in GA-impacted colonies. Relatively high variation in metabolomic profiles among coral samples of the same treatment (i.e., inter-colony variation) confounded data interpretation, however, analyses of paired GA and unaffected samples identified 73 features that differed between these respective metabolome types. These features were largely annotated as unknowns, but 1-methylnicotinamide and trigonelline were found to be elevated in GA samples, while betaine, glycine, and histamine were lower in GA samples. Pathway analyses indicate decreased choline oxidation in GA samples, making this a pathway of interest for future targeted studies. Collectively, our results provide unique insights into GA pathophysiology by showing these lesions alter both the absolute and relative metabolism of affected colonies and by identifying features (metabolites and unknowns) and metabolic pathways of interest in GA pathophysiology going forward.

     

Benthic microalgae in coral reef sediments of the southern Great Barrier Reef,Australia

The abundance and productivity of benthic microalgae in coral reef sediments are poorly known compared with other, more conspicuous (e.g. coral zooxanthellae, macroalgae) primary producers of coral reef habitats. A survey of the distribution, biomass, and productivity of benthic microalgae on a platform reef flat and in a cross-shelf transect in the southern Great Barrier Reef indicated that benthic microalgae are ubiquitous, abundant (up to 995.0 mg chlorophyll (chl) a m^–2), and productive (up to 110 mg O₂ m^–2 h^–1) components of the reef ecosystem. Concentrations of benthic microalgae, expressed as chlorophyll a per surface area, were approximately 100-fold greater than the integrated water column concentrations of microalgae throughout the region. Benthic microalgal biomass was greater on the shallow water platform reef than in the deeper waters of the cross-shelf transect. In both areas the benthic microalgal communities had a similar composition, dominated by pennate diatoms, dinoflagellates, and cyanobacteria. Benthic microalgal populations were potentially nutrient-limited, based on responses to nitrogen and phosphorus enrichments in short-term (7-day) microcosm experiments. Benthic microalgal productivity, measured by O₂ evolution, indicated productive communities responsive to light and nutrient availability. The benthic microalgal concentrations observed (92–995 mg chl a m^–2) were high relative to other reports, particularly compared with temperate regions. This abundance of productive plants in both reef and shelf sediments in the southern Great Barrier Reef suggests that benthic microalgae are key components of coral reef ecosystems.Communicated by Environmental Editor, B.C. Hatcher     

Feeding experiment using uniformly 13C-labeled α-linolenic acid supports the involvement of the decarboxylation mechanism to produce cis-jasmone in Lasiodiplodia theobromae

ABSTRACT

In our previous report, it was found that Lasiodiplodia theobromae produced cis-jasmone via partially utilizing the biosynthetic pathway of JA. A feeding experiment using uniformly ¹³C-labeled α-linolenic acid, which was added to the culture media of the fungus, strongly supported that the fungus produced CJ via the decarboxylation step of the biosynthetic pathway.     

Coral bleaching is linked to the capacity of the animal host to supply essential metals to the symbionts

Massive coral bleaching events result in extensive coral loss throughout the world. These events are mainly caused by seawater warming, but are exacerbated by the subsequent decrease in nutrient availability in surface waters. It has therefore been shown that nitrogen, phosphorus or iron limitation contribute to the underlying conditions by which thermal stress induces coral bleaching. Generally, information on the trophic ecology of trace elements (micronutrients) in corals, and on how they modulate the coral response to thermal stress is lacking. Here, we demonstrate for the first time that heterotrophic feeding (i.e. the capture of zooplankton prey by the coral host) and thermal stress induce significant changes in micro element concentrations and isotopic signatures of the scleractinian coral Stylophora pistillata. The results obtained first reveal that coral symbionts are the major sink for the heterotrophically acquired micronutrients and accumulate manganese, magnesium and iron from the food. These metals are involved in photosynthesis and antioxidant protection. In addition, we show that fed corals can maintain high micronutrient concentrations in the host tissue during thermal stress and do not bleach, whereas unfed corals experience a significant decrease in copper, zinc, boron, calcium and magnesium in the host tissue and bleach. In addition, the significant increase in δ⁶⁵Cu and δ⁶⁶Zn signature of symbionts and host tissue at high temperature suggests that these isotopic compositions are good proxy for stress in corals. Overall, present findings highlight a new way in which coral heterotrophy and micronutrient availability contribute to coral resistance to global warming and bleaching.     

Differential protein abundance during the first month of regeneration of the Caribbean star coral Montastraea cavernosa

It is critical to determine the methods by which coral colonies regenerate tissue lost to physical injury as they provide the physical structure of coral reef systems. To explore regeneration, circular lesions (12 mm diameter × 3 mm depth) were created in the fall of 2014 on 124 Montastraea cavernosa colonies located in the coastal waters of Grenada and Carriacou (10–12 m depth). Coral regeneration was documented at weekly intervals for 28 days. Repeated measures ANOVA on estimated weekly coral regeneration rates showed that island (p = 0.024) and colony colour (p = 0.024) were the only factors significantly affecting lesion regeneration. Mean rate of lesion closure during the first 28 days was approximately 2.8 mm² d⁻¹. Four identical circular lesions were created on 30 M. cavernosa colonies (Carriacou, 10–12 m depth) in the fall of 2015. One representative lesion created on each coral colony was re-sampled at each of 14, 21, and 32 or 33 days following injury, and coral tissue was flash-frozen. Tissues from 10 normally pigmented brown colonies were selected for proteomic analysis using tandem mass tags. The initial polyp sample, the day 14, and the final samples were used to quantify the difference in protein abundance as the lesions healed. In the tissue samples 6419 peptides were reliably identified, which corresponded to 906 unique proteins. During the first month of regeneration, 111 proteins were differentially abundant (p < 0.05) on at least one timepoint and of these, 11 were associated with regeneration. An additional 14 proteins were also identified that were differentially abundant (p < 0.05) and were associated with inflammation or antioxidant activity. This work demonstrates, for the first time, the differential abundance of proteins associated with regeneration in a scleractinian coral.