源于微生物的海鞘天然产物

海洋动物是具有生物活性海洋天然产物的重要来源。海鞘中含有丰富的微生物类群,如细菌、放线菌、真菌和蓝细菌。越来越多的直接或间接证据表明,一些从海鞘中分离的天然产物并不是海鞘本身产生的,而是由其共生微生物产生的。本文对近些年来的海鞘天然产物的微生物来源的研究方法进行综述,包括可培养细菌的分离、不可培养细菌的粗提物检测、宏基因组学、全基因组测序等直接方法,以及化合物结构比对的间接方法。通过对海鞘-微生物共生体中天然产物生物合成来源的研究,不仅可以从根本上解决动物药源的问题,而且可为研究海鞘与微生物共生关系提供有力证据。     

Production of isoprenoid pharmaceuticals by engineered microbes

Throughout human history, natural products have been the foundation for the discovery and development of therapeutics used to treat diseases ranging from cardiovascular disease to cancer. Their chemical diversity and complexity have provided structural scaffolds for small-molecule drugs and have consistently served as inspiration for medicinal design. However, the chemical complexity of natural products also presents one of the main roadblocks for production of these pharmaceuticals on an industrial scale. Chemical synthesis of natural products is often difficult and expensive, and isolation from their natural sources is also typically low yielding. Synthetic biology and metabolic engineering offer an alternative approach that is becoming more accessible as the tools for engineering microbes are further developed. By reconstructing heterologous metabolic pathways in genetically tractable host organisms, complex natural products can be produced from inexpensive sugar starting materials through large-scale fermentation processes. In this Perspective, we discuss ongoing research aimed toward the production of terpenoid natural products in genetically engineered Escherichia coli and Saccharomyces cerevisiae.     

Isolation of the β-galactosphingolipid coniferoside using a tumor cell proteome reverse affinity protocol

New approaches are vital to the development of marine natural products (MNP) as therapeutic leads. One of the more time consuming aspects of MNP research arises in the connection between structure and function. Here, we describe an isolation protocol that adapts tumor cell proteomes as a vehicle for MNP isolation therein uniting structural and functional analysis. Application of this method to extracts of the sponge Agelas conifera led to the isolation of a unique poly-hydroxybutyrated β-galactosphingolipid, coniferoside.     

Indirect Evidence that Flying Foxes Track Food Resources Among Islands in a Pacific Archipelago

Although flying foxes (fruit bats in the genus Pteropus ) in continental forests often fly between scattered resources, little is known about their ranging behavior among islands. The inhospitable water matrix that surrounds the food patches (islands) in archipelagos may prevent flying foxes from tracking resources as efficiently as their counterparts on larger landmasses do. Our aim in this study was to determine whether the abundance of foraging flying foxes ( Pteropus tonganus ) reflected food availability on islands in the Vava'u archipelago of Tonga, regardless of island size and isolation. Overall, food availability was the strongest determinant of flying fox abundance, and spatial aspects of the islands (land area within 10 km) had only a small influence. Food availability appears to regulate flying fox abundance only when food resources are low, but when food sources are plentiful, flying fox abundance may be high or low. These results provide indirect evidence that flying foxes are able to track food resources efficiently in an archipelago, and the water matrix that surrounds the food patches (islands) is not a strong deterrent for foraging animals.     

Campylobacter bacteriophages and bacteriophage therapy

Members of the genus Campylobacter are frequently responsible for human enteric disease with occasionally very serious outcomes. Much of this disease burden is thought to arise from consumption of contaminated poultry products. More than 80% of poultry in the UK harbour Campylobacter as a part of their intestinal flora. To address this unacceptably high prevalence, various interventions have been suggested and evaluated. Among these is the novel approach of using Campylobacter-specific bacteriophages, which are natural predators of the pathogen. To optimize their use as therapeutic agents, it is important to have a comprehensive understanding of the bacteriophages that infect Campylobacter, and how they can affect their host bacteria. This review will focus on many aspects of Campylobacter-specific bacteriophages including: their first isolation in the 1960s, their use in bacteriophage typing schemes, their isolation from the different biological sources and genomic characterization. As well as their use as therapeutic agents to reduce Campylobacter in poultry their future potential, including their use in bio-sanitization of food, will be explored. The evolutionary consequences of naturally occurring bacteriophage infection that have come to light through investigations of bacteriophages in the poultry ecosystem will also be discussed.     

Making small molecules in plants: A chassis for synthetic biology-based production of plant natural products

Plant natural products have been extensively exploited in food,medicine,flavor,cosmetic,renewable fuel,and other industrial sectors.Synthetic biology has recently emerged as a promising means for the cost-effective and sustainable production of natural products.Compared with engineering microbes for the production of plant natural products,the potential of plants as chassis for producing these compounds is underestimated,largely due to challenges encountered in engineering plants.Knowledge in pl...     

Screening concepts,characterization and structural analysis of microbial-derived bioactive lipopeptides: a review

Lipopeptide biosurfactants are surface active biomolecules that are produced by a variety of microorganisms. Microbial lipopeptides have gained the interest of microbiologists, chemists and biochemists for their high biodiversity as well as efficient action, low toxicity and good biodegradability in comparison to synthetic counterparts. In this report, we review methods for the production, isolation and screening, purification and structural characterization of microbial lipopeptides. Several techniques are currently available for each step, and we describe the most commonly utilized and recently developed techniques in this review. Investigations on lipopeptide biosurfactants in natural products require efficient isolation techniques for the characterization and evaluation of chemical and biological properties. A combination of chromatographic and spectroscopic techniques offer opportunities for a better characterization of lipopeptide structures, which in turn can lead to the application of lipopeptides in food, pharmaceutical, cosmetics, agricultural and bioremediation industries.     

Deep sequencing of non-ribosomal peptide synthetases and polyketide synthases from the microbiomes of Australian marine sponges

The biosynthesis of non-ribosomal peptide and polyketide natural products is facilitated by multimodular enzymes that contain domains responsible for the sequential condensation of amino and carboxylic subunits. These conserved domains provide molecular targets for the discovery of natural products from microbial metagenomes. This study demonstrates the application of tag-encoded FLX amplicon pyrosequencing (TEFAP) targeting non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes as a method for determining the identity and diversity of natural product biosynthesis genes. To validate this approach, we assessed the diversity of NRPS and PKS genes within the microbiomes of six Australian marine sponge species using both TEFAP and metagenomic whole-genome shotgun sequencing approaches. The TEFAP approach identified 100 novel ketosynthase (KS) domain sequences and 400 novel condensation domain sequences within the microbiomes of the six sponges. The diversity of KS domains within the microbiome of a single sponge species Scopalina sp. exceeded that of any previously surveyed marine sponge. Furthermore, this study represented the first to target the condensation domain from NRPS biosynthesis and resulted in the identification of a novel condensation domain lineage. This study highlights the untapped potential of Australian marine sponges for the isolation of novel bioactive natural products. Furthermore, this study demonstrates that TEFAP approaches can be applied to functional genes, involved in natural product biosynthesis, as a tool to aid natural product discovery. It is envisaged that this approach will be used across multiple environments, offering an insight into the biological processes that influence the production of secondary metabolites.     

Analysis of food allergen structures and development of foods for allergic patients

Food allergy is one of the important health problems, and countermeasures are socially required. We have been undertaking studies on wheat allergens and their epitopes, and have developed a method for producing hypoallergenic wheat flour by enzymatic modification. The hypoallergenic products are now provided to patients. More noteworthy, by taking hypoallergenic cupcakes over a long period, more than half of patients are hyposensitized and become able to eat normal wheat products. This suggests that the hypoallergenic wheat flour can act as anti-allergenic via allergen-specific immunotolerance. This series of studies was followed by expansive research on food allergy: analysis of epitopes of bovine serum albumin (the major beef allergen), isolation and identification of inhibitory peptides for allergen absorption at the intestine, evaluation of hesperetin as an inhibitor of degranulation of mast cells, and the development of PCR detection methods for verifying allergen labeling and for identifying hidden allergic ingredients in processed foods.     

Hydrolytic breakdown of lactoferricin by lactic acid bacteria

Lactoferricin is a 25-amino acid antimicrobial peptide fragment that is liberated by pepsin digestion of lactoferrin present in bovine milk. Along with its antibacterial properties, lactoferricin has also been reported to have immunostimulatory, antiviral, and anticarcinogenic effects. These attributes provide lactoferricin and other natural bioactive peptides with the potential to be functional food ingredients that can be used by the food industry in a variety of applications. At present, commercial uses of these types of compounds are limited by the scarcity of information on their ability to survive food processing environments. We have monitored the degradation of lactoferricin during its incubation with two types of lactic acid bacteria used in the yogurt-making industry, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, with the aim of assessing the stability of this milk protein-derived peptide under simulated yogurt-making conditions. Analysis of the hydrolysis products isolated from these experiments indicates degradation of this peptide near neutral pH by lactic acid bacteria-associated peptidases, the extent of which was influenced by the bacterial strain used. However, the data also showed that compared to other milk-derived bioactive peptides that undergo complete degradation under these conditions, the 25-amino acid lactoferricin is apparently more resistant, with approximately 50% of the starting material remaining after 4 h of incubation. These findings imply that lactoferricin, as a natural milk protein-derived peptide, has potential applications in the commercial production of yogurt-like fermented dairy products as a multi-functional food ingredient.     

Carbon/nitrogen relationships of fungus culture media

Summary and Conclusions Four basic nutrient types are described and discussed in special reference to the ratios between carbon source materials and nitrogen source materials. These include habitat, natural nutrients, semisynthetic nutrients and synthetic nutrients. Special emphasis is placed on the use of such nutrients in agar media for the primary isolation of fungi from various types of habitat materials such as soils and sewage.It is suggested that a basic C:N ratio for such nutrient agars lies at about 9 or 12 to 1, that increasing this ratio tends to overenrich the culture thus encouraging the formation of intracellular or extracellular carbon-carrying compounds, and that decreasing the ratio tends to encourage the formation of nitrogen-carrying storage products. Both conditions tend to produce staling substances more rapidly, tend to decrease discreteness of colonies, tend to produce more diffuse, less easily counted growth, and tend to develop less readily identifiable colonies.It is implied that if a C:N ratio more or less approximating that found in an agar containing plant parts, or limited nutrients plus plant parts, were used in a medium designed for primary isolation of fungi from natural materials, such a medium would have many advantages over currently popular media used for this purpose.     

Influence of aquatic microbiota on the survival in water of the human and eel pathogen Vibrio vulnificus serovar E

The eel and human pathogen Vibrio vulnificus serovar E (biotype 2) is seldom isolated from natural waters, although it can survive in sterilized artificial seawater microcosms for years. The main objective of the present study was to investigate whether aquatic microbiota can limit its survival and recovery from water samples. A set of preliminary experiments of survival in microcosms containing natural seawater and water from eel farms showed that the persistence of this pathogen was mainly controlled by grazing, and secondarily by bacterial competition. The bacterial competition was further analysed in artificial seawater microcosms co-inoculated with selected virulent serovar E (VSE) strains and potential competitors. Competitors included V. vulnificus biotype 1 isolates and strains of selected species that can grow on the selective media designed for V. vulnificus isolation from water samples. Evidences of bacterial competition that was detrimental for VSE recovery were recorded. Thus, some species produced a deleterious effect on VSE strains under starvation, and others were able to use the resources more efficiently under nutrient input. These results suggest that an overgrowth of more efficient competitor bacteria in conventional media used for isolation of V. vulnificus could mask the recovery of VSE strains and explain the scarcity of reports on the isolation of this human and eel pathogen from natural waters.     

Body size evolution simultaneously creates and collapses species boundaries in a clade of scincid lizards

Speciation is generally viewed as an irreversible process, although habitat alterations can erase reproductive barriers if divergence between ecologically differentiated species is recent. Reversed speciation might also occur if geographical contact is established between species that have evolved the same reproductive isolating barrier in parallel. Here, we demonstrate a loss of intrinsic reproductive isolation in a clade of scincid lizards as a result of parallel body size evolution, which has allowed for gene flow where large-bodied lineages are in secondary contact. An mtDNA phylogeny confirms the monophyly of the Plestiodon skiltonianus species complex, but rejects that of two size-differentiated ecomorphs. Mate compatibility experiments show that the high degree of body size divergence imposes a strong reproductive barrier between the two morphs; however, the strength of the barrier is greatly diminished between parallel-evolved forms. Since two large-bodied lineages are in geographical contact in the Sierra Nevada Mountains of California, we were also able to test for postzygotic isolation under natural conditions. Analyses of amplified fragment length polymorphisms show that extensive gene exchange is occurring across the contact zone, resulting in an overall pattern consistent with isolation by distance. These results provide evidence of reversed speciation between clades that diverged from a common ancestor more than 12Myr ago.     

2 Protein Folding and its Implications for the Production of Recombinant Proteins

Abstract

Humans have utilised plant derived natural products as medicines for millenia. Moreover, many contemporary pharmaceuticals are also natural products or derivatives thereof. However, the full potential of these compounds remains to be exploited because often they are: complex and difficult to synthesise; found in low quantities; produced by undomesticated and sometimes rare plants; and, their synthesis is routinely influenced by weather conditions. Potentially, the in vitro culture of cells from the corresponding plant species could circumvent some of these problems but the growth of plant cells on an industrial scale is also problematic. The recent isolation and culture of cambial meristematic cells (CMCs), stem cells which ordinarily generate the plant vasculature, may now provide a key platform technology to help realise the full potential of plant natural products.     

Listeria monocytogenes isolates from foods and humans form distinct but overlapping populations

A total of 502 Listeria monocytogenes isolates from food and 492 from humans were subtyped by EcoRI ribotyping and PCR-restriction fragment length polymorphism analysis of the virulence gene hly. Isolates were further classified into genetic lineages based on subtyping results. Food isolates were obtained through a survey of selected ready-to-eat food products in Maryland and California in 2000 and 2001. Human isolates comprised 42 isolates from invasive listeriosis cases reported in Maryland and California during 2000 and 2001 as well as an additional 450 isolates from cases that had occurred throughout the United States, predominantly from 1997 to 2001. Assignment of isolates to lineages and to the majority of L. monocytogenes subtypes was significantly associated with the isolate source (food or human), although most subtypes and lineages included both human and food isolates. Some subtypes were also significantly associated with isolation from specific food types. Tissue culture plaque assay characterization of the 42 human isolates from Maryland and California and of 91 representative food isolates revealed significantly higher average infectivity and cell-to-cell spread for the human isolates, further supporting the hypothesis that food and human isolates form distinct populations. Combined analysis of subtype and cytopathogenicity data showed that strains classified into specific ribotypes previously linked to multiple human listeriosis outbreaks, as well as those classified into lineage I, are more common among human cases and generate larger plaques than other subtypes, suggesting that these subtypes may represent particularly virulent clonal groups. These data will provide a framework for prediction of the public health risk associated with specific L. monocytogenes subtypes.     

Isolating structures of gerbils’ digestive tract (Gerbillidae, <Emphasis Type="Italic">Rhombomys,Meriones</Emphasis>) and their functional significance

The morphology of the digestive tract of three species of gerbils characterized by different food specialization—Rhombomys opimus, Meriones tamariscinus and M. meridianus—is considered. The correlation between areas of glandular and cornified epithelium is variable and depends greatly on the type of stomach filling and distribution of food mass in it. The bordering fold and ileocecal junction is not only able to isolate the forestomach and caecum significantly but also may be a mechanism of regulation of food movement through the digestive tract. The close location of ileum’s opening to the caecum and its outlet to the colon, as well as isolation of caecum’s cavity by a circular fold, could provide direct food transit from the ileum to the colon.     

Genetic evidence for natural product-mediated plant-plant allelopathy in rice (Oryza sativa)

? There is controversy as to whether specific natural products play a role in directly mediating antagonistic plant-plant interactions - that is, allelopathy. If proved to exist, such phenomena would hold considerable promise for agronomic improvement of staple food crops such as rice (Oryza sativa). ? However, while substantiated by the presence of phytotoxic compounds at potentially relevant concentrations, demonstrating a direct role for specific natural products in allelopathy has been difficult because of the chemical complexity of root and plant litter exudates. This complexity can be bypassed via selective genetic manipulation to ablate production of putative allelopathic compounds, but such an approach previously has not been applied. ? The rice diterpenoid momilactones provide an example of natural products for which correlative biochemical evidence has been obtained for a role in allelopathy. Here, we apply reverse genetics, using knock-outs of the relevant diterpene synthases (copalyl diphosphate synthase 4 (OsCPS4) and kaurene synthase-like 4 (OsKSL4)), to demonstrate that rice momilactones are involved in allelopathy, including suppressing growth of the widespread rice paddy weed, barnyard grass (Echinochloa crus-galli). ? Thus, our results not only provide novel genetic evidence for natural product-mediated allelopathy, but also furnish a molecular target for breeding and metabolic engineering of this important crop plant.     

The comeback of hirudin--an old-established anticoagulant agent

Early studies dating back to 1884 revealed that extracts from medicinal leeches contain a substance which is able to prevent blood from clotting. Since our successful isolation of hirudin, the pure anticoagulant substance, in the late 1950s and its characterization as a selective thrombin inhibitor with polypeptide structure, hirudin preparations have been employed for diagnostic and scientific uses in haemostaseology. As early as 25 years ago we have shown in experimental pharmacotoxicological studies that hirudin is an anticoagulant of high quality. The antithrombotic effect of hirudin was demonstrated in several thrombosis models. But the clinical use of hirudin remained limited since it was not available in adequate amounts for therapeutic purposes. One hundred years after its discovery there is a renewed interest in this naturally occurring thrombin inhibitor. Advanced methods of peptide isolation and genetic engineering are about to provide sufficient quantities of hirudin in purified form. This prompted us to resume our investigations in hirudin and to represent new experimental and clinical pharmacological studies with natural hirudin prepared from medicinal leeches and genetically engineered recombinant hirudin, thus appreciating the comeback of hirudin into the focus of interest.     

Metabolomic Profile of the Genus Inula

Plants have a long history as therapeutics in the treatment of human diseases and have been used as source of medicines for ages. Searching for new biologically active natural products, many plants and herbs are screened for natural products with pharmacological activities. In this field, the genus Inula, which comprises more than 100 species, several of them being used in traditional medicine, is very important, especially due to the finding that several of the isolated pure secondary metabolites proved to possess important biological activities. Inula species have been reported as rich sources of sesquiterpene lactones, including eudesmanes, germacranes, guaianes, and dimeric structures, and since 2006 ca. 400 secondary metabolites, including more than 100 new natural products, some of them with relevant pharmacological activities, have been identified. Herein, we critically compile and update the information regarding the types of secondary metabolites found in the genus Inula and the progress in their isolation.