Synthetic biology and metabolic engineering of actinomycetes for natural product discovery

Actinomycetes are one of the most valuable sources of natural products with industrial and medicinal importance. After more than half a century of exploitation, it has become increasingly challenging to find novel natural products with useful properties as the same known compounds are often repeatedly re-discovered when using traditional approaches. Modern genome mining approaches have led to the discovery of new biosynthetic gene clusters, thus indicating that actinomycetes still harbor a huge unexploited potential to produce novel natural products. In recent years, innovative synthetic biology and metabolic engineering tools have greatly accelerated the discovery of new natural products and the engineering of actinomycetes. In the first part of this review, we outline the successful application of metabolic engineering to optimize natural product production, focusing on the use of multi-omics data, genome-scale metabolic models, rational approaches to balance precursor pools, and the engineering of regulatory genes and regulatory elements. In the second part, we summarize the recent advances of synthetic biology for actinomycetal metabolic engineering including cluster assembly, cloning and expression, CRISPR/Cas9 technologies, and chassis strain development for natural product overproduction and discovery. Finally, we describe new advances in reprogramming biosynthetic pathways through polyketide synthase and non-ribosomal peptide synthetase engineering. These new developments are expected to revitalize discovery and development of new natural products with medicinal and other industrial applications.     

Bird Assemblages in Anthropogenic Habitats: Identifying a Suitability Gradient for Native Species in the Atlantic Forest

Traditional approaches to the study of species persistence in fragmented landscapes generally consider a binary classification of habitat being suitable or unsuitable; however, the range of human‐modified habitats within a region may offer a gradient of habitat suitability (or conservation value) for species. We identified such a gradient by comparing bird assemblages among contrasting land uses (pine plantations of different age, annual crops, clear cuts and cattle pastures) in the Upper Parana Atlantic forest. Bird assemblages and vegetation structure were characterized in an extensive area of 4400 km² in Argentina and Paraguay during the breeding seasons of 2005–2010. Similarity of bird assemblages between anthropogenic habitats and the native forest and the proportion of forest species increased with vegetation vertical structure, while the proportion of open‐area species decreased. As a consequence, mature tree plantations were the most suitable habitats for forest species and were mainly used by frugivores and bark insectivores. In contrast, open habitats were the least suitable habitat for forest species and were used primarily by insectivores. Human‐created habitats that are structurally complex can be used by a subset of forest species, and may improve functional connectivity and mitigate edge effects. The conservation of large tracks of native forests, however, is critical for the long‐term persistence of the entire bird assemblage, especially for native forest dependent species.     

Community of small mammals along an elevational gradient in Biological Reserve of Serra do Japi,municipality of Jundiaí‐SP,Brazil

Studies of elevational gradients in forests are particularly interesting for the considerable differences that can be observed over short distances, such as in vegetation and temperature. Different taxonomic groups display varying types of distribution patterns along elevational gradients, with unimodal distribution being recognised as the most common pattern. The distribution of species can be affected by a range of factors that include, biotic, spatial, climatic, historic and energetic. Small mammals represent an ideal model for studies about distribution and habitat use as they can be highly abundant, tend to have different diets and use space differently. The aims of this study are to build a comprehensive understanding of the community of small mammals of the Biological Reserve of Serra do Japi and to explore its distribution pattern along elevational gradients. We investigated the influence of biomass of arthropods, fruits and seeds and percentage of ground cover, canopy cover and vertical vegetation at richness and abundance of small mammals at three different elevations. To accomplish this, we used seventy‐two pitfall traps of 63 L to capture small mammals and distributed them equally across three elevations defined as low (880–899 m), intermediate (1046–1089 m) and high (1170–1189 m). Each elevation had three lines or replicas of traps. Throughout the study, we captured one hundred and fourteen individuals belonging to eleven species of small mammals. The presence of rare and endemic species demonstrates the importance of conservation and maintenance of the Biological Reserve of Serra do Japi. In regard to the distribution of species, despite the short gradient range, we found a unimodal pattern and a positive correlation between ground cover (fallen twigs and branches up to 1 m high) and richness and abundance of small mammals. More ground cover can reduce the effects of competition and predation on small mammals’ communities. Abstract in Portuguese is available with online material.     

Sutural bone frequency in synostotic rabbit crania

This study tests the hypothesis that crania with synostosed sutures will have a significantly higher incidence of calvarial sutural bones than normal crania. Sutural bones were counted in seven calvarial sutures and compared among four groups of adult New Zealand white rabbit skulls: normal in-colony (NI) controls (N = 14), normal out-colony (NO) controls (N = 12), skulls with familial delayed onset (DO) coronal synostosis (N = 25), and skulls with experimentally immobilized coronal sutures (EI) (N = 20). Comparisons among groups were made with a Kruskal-Wallis one-way ANOVA and between groups with a Mann-Whitney U-test, using a Bonferroni correction for multiple comparisons. Significant differences (P > 0.05) were noted only in the coronal and sagittal sutures, with EI crania having the greatest number of coronal sutural bones; between group differences were undetectable for sagittal sutural bones. A post hoc two-sample binomial test for equal proportions showed that the distribution of coronal sutural bones among individuals across groups was even, while the distribution of sagittal sutural bones was significantly higher in EI crania. These results suggest that altered sutural forces of the calvaria contribute to an increased occurrence of sutural bones. However, the influence of inheritance on increased occurrence of sutural bones cannot be discounted, as reflected in the equivalent number of individuals across groups that possessed coronal sutural bones. Am J Phys Anthropol 102:555–563, 1997. © 1997 Wiley-Liss, Inc.     

In vitro rearing ofExorista larvarum on tissue culture-based diets

Exorista (=Tachina) larvarum (L.) (Diptera, Tachinidae), a polyphagous parasitoid that attacksLymantria dispar L. andHyphantria cunea (Drury), was rearedin vitro from egg to adult on four tissue culture media-based diets (TMM-FH, SCHNEIDER'S, EX-CELL 400, and SF-900). The kind of tissue culture media in the diets did not influence the adult yield (34 to 55%) and puparium weight (26–27 mg). Adult yield and the puparium weight ofE. larvarum developed on TNM-FH and SCHNEIDER'S-based diets containing different amounts ofGalleria mellonella pupal extract (PE) (0, 1.25, 2.5 and 5%), were lower on diets without PE. In diets without PE development times from oviposition to adult emergence, were shorter on TNM-FH (19 days) than on SCHNEIDER'S-based diet (25–26 days). The adults that developed on artificial diets were able to parasitize the factitious hostG. mellonella and produce viable progeny. The results demonstrate thatE. larvarum is the most promising parasitoid ever studied forin vitro mass production.     

Agriculture and herbivorous waterfowl: a review of the scientific basis for improved management

Swans, geese and some ducks (Anatidae) are obligate herbivores, many are important quarry species and all contribute to a variety of ecosystem services. Population growth and shifting ranges have led to increasing proximity to man and thus increasing conflicts. We review and synthesize the role of these birds as herbivores on agricultural land (cropland, rotational grassland and pasture) and other terrestrial habitats where conflict with human interests may occur. A bibliographic analysis of peer‐reviewed papers (N = 359) shows that publication activity peaked in 1991–2000 in North America and 2000–2010 in Europe, and has decreased since. Taxonomic and geographical biases are obvious in research to date: Snow Goose Chen caerulescens was the most studied species (N = 98), and Canada Branta canadensis, Barnacle B. leucopsis and Brent geese B. bernicla all featured in more than 40 studies; most studies originated in northwest Europe or North America, very few have been carried out in Asia and European Russia. On the basis of nutrient/energy budgets of herbivorous waterfowl, it is evident that dense single‐species crops (such as rotational grassland, early‐growth cereals and root crops) and spilled grain in agricultural landscapes offer elevated energetic and nutritional intake rates of food of higher quality compared to natural or semi‐natural vegetation. Hence, although affected by seasonal nutritional demands, proximity to roost, field size, disturbance levels, access to water, food depletion and snow cover, agricultural landscapes tend to offer superior foraging opportunities over natural habitats, creating potential conflict with agriculture. Herbivorous waterfowl select for high protein, soluble carbohydrate and water content, high digestibility as well as low fibre and phenolic compounds, but intake rates from grazing varied with goose body and bill morphology, creating species‐specific loci for conflict. Crop damage by trampling and puddling has not been demonstrated convincingly, nor do waterfowl faeces deter grazing stock, but where consumption of crops evidently reduces yields this causes conflict with farmers. Studies show that it is difficult and expensive to assess the precise impacts of waterfowl feeding on yield loss because of other sources of variation. However, less damage has been documented from winter grazing compared to spring grazing and yield loss after spring grazing on grassland appears more pronounced than losses on cereal fields. Although yield losses at national scales are trivial, individual farmers in areas of greatest waterfowl feeding concentrations suffer disproportionately, necessitating improved solutions to conflict. Accordingly, we review the efficacy of population management, disturbance, provision of alternative feeding areas, compensation and large‐scale stakeholder involvement and co‐management as options for resolving conflict based on the existing literature and present a framework of management advice for the future. We conclude with an assessment of the research needs for the immediate future to inform policy development, improve management of waterfowl populations and reduce conflict with agriculture.     

Widespread supplementary feeding in domestic gardens explains the return of reintroduced Red Kites Milvus milvus to an urban area

Reintroductions are commonly used to mitigate biodiversity loss. One prominent example is that of the Red Kite Milvus milvus, a charismatic raptor of conservation concern. This species has been reintroduced across the UK over the last 25 years following its near extinction after centuries of persecution. The species was not expected to recolonize urban areas; its historical association with human settlements is attributed to scavenging on human waste and refuse, a resource now greatly reduced on the streets of modern European cities. However, the species has become a common daytime visitor to a large conurbation centred on the town of Reading, southern England, approximately 20 km from the first English reintroduction site. Given a near‐absence of breeding and roost sites, we investigated foraging opportunities and habitat associations that might explain use by Red Kites of this urban area. Surveys of discarded human foods and road‐kill suggested that these could support at most 13–29 Kites per day. Face‐to‐face surveys of a cross‐section of residents revealed that 4.5% (equivalent to 4349 households) provided supplementary food for Red Kites in their gardens. Using estimates of per‐household resource provision from another study, we calculated that this is potentially sufficient to feed 142–320 Kites, a substantial proportion of the total estimated to visit the conurbation each day (between 140 and 440). Road transects found positive associations between Red Kites and residential areas. We suggest that the decision made by thousands of householders to provide supplementary food for Red Kites in their gardens is the primary factor explaining their daytime abundance in this urban area.     

Evolution of the premaxillary fraenum and substratum in snubnose darters and allies (Percidae: Etheostoma)

Darters (Percidae: Etheostomatinae), a species‐rich group of North American freshwater fishes, vary in the presence of a premaxillary fraenum, a strip of skin that connects the premaxillary bones to the snout, and it is hypothesized that this trait is a trophic adaptation to particular substrata. Ancestral state reconstructions and analyses of phylogenetic associations between presence of the premaxillary fraenum and preferred stream substratum were conducted in a clade of closely related darters (snubnose darters and allies) that vary in morphology and habitat preferences. The most recent common ancestor of this clade was inferred to possess a fraenum and to inhabit rocky substrata, consistent with previous hypotheses, but a significant correlation between fraenum presence and substratum type across the phylogeny was not found.