Project description:Filamentous fungi are highly productive cell factories, often used in industry for the production of enzymes and small bioactive compounds. Recent years have seen an increasing number of synthetic-biology-based applications in fungi, emphasizing the need for a synthetic biology toolkit for these organisms. Here we present a collection of 96 genetic parts, characterized in Penicillium or Aspergillus species, that are compatible and interchangeable with the Modular Cloning system. The toolkit contains natural and synthetic promoters (constitutive and inducible), terminators, fluorescent reporters, and selection markers. Furthermore, there are regulatory and DNA-binding domains of transcriptional regulators and components for implementing different CRISPR-based technologies. Genetic parts can be assembled into complex multipartite assemblies and delivered through genomic integration or expressed from an AMA1-sequence-based, fungal-replicating shuttle vector. With this toolkit, synthetic transcription units with established promoters, fusion proteins, or synthetic transcriptional regulation devices can be more rapidly assembled in a standardized and modular manner for novel fungal cell factories.
Project description:Fungal synthetic biology is a rapidly expanding field that aims to optimize the biotechnological exploitation of fungi through the generation of standard, ready-to-use genetic elements, and universal syntax and rules for contributory use by the fungal research community. Recently, an increasing number of synthetic biology toolkits have been developed and applied to filamentous fungi, which highlights the relevance of these organisms in the biotechnology field. The FungalBraid (FB) modular cloning platform enables interchangeability of DNA parts with the GoldenBraid (GB) platform, which is designed for plants, and other systems that are compatible with the standard Golden Gate cloning and syntax, and uses binary pCAMBIA-derived vectors to allow Agrobacterium tumefaciens-mediated transformation of a wide range of fungal species. In this study, we have expanded the original FB catalog by adding 27 new DNA parts that were functionally validated in vivo. Among these are the resistance selection markers for the antibiotics phleomycin and terbinafine, as well as the uridine-auxotrophic marker pyr4. We also used a normalized luciferase reporter system to validate several promoters, such as PpkiA, P7760, Pef1α, and PafpB constitutive promoters, and PglaA, PamyB, and PxlnA inducible promoters. Additionally, the recently developed dCas9-regulated GB_SynP synthetic promoter collection for orthogonal CRISPR activation (CRISPRa) in plants has been adapted in fungi through the FB system. In general, the expansion of the FB catalog is of great interest to the scientific community since it increases the number of possible modular and interchangeable DNA assemblies, exponentially increasing the possibilities of studying, developing, and exploiting filamentous fungi.
Project description:High-frequency, in-situ monitoring provides large environmental datasets. These datasets will likely bring new insights in landscape functioning and process scale understanding. However, tailoring data analysis methods is necessary. Here, we detach our analysis from the usual temporal analysis performed in hydrology to determine if it is possible to infer general rules regarding hydrochemistry from available large datasets. We combined a 2-year in-stream nitrate concentration time series (time resolution of 15 min) with concurrent hydrological, meteorological and soil moisture data. We removed the low-frequency variations through low-pass filtering, which suppressed seasonality. We then analyzed the high-frequency variability component using Pareto Density Estimation, which to our knowledge has not been applied to hydrology. The resulting distribution of nitrate concentrations revealed three normally distributed modes: low, medium and high. Studying the environmental conditions for each mode revealed the main control of nitrate concentration: the saturation state of the riparian zone. We found low nitrate concentrations under conditions of hydrological connectivity and dominant denitrifying biological processes, and we found high nitrate concentrations under hydrological recession conditions and dominant nitrifying biological processes. These results generalize our understanding of hydro-biogeochemical nitrate flux controls and bring useful information to the development of nitrogen process-based models at the landscape scale.
Project description:Volatile organic compounds (VOCs), a bouquet of chemical compounds released by all life forms, play essential roles in trophic interactions. VOCs can facilitate a large number of interactions with different organisms belowground. VOCs-regulated plant-plant or plant-insect interaction both below and aboveground has been reported extensively. Nevertheless, there is little information about the role of VOCs derived from soilborne pathogenic fungi and beneficial fungi, particularly mycorrhizae, in influencing plant performance. In this review, we show how plant VOCs regulate plant-soilborne pathogenic fungi and beneficial fungi (mycorrhizae) interactions. How fungal VOCs mediate plant-soilborne pathogenic and beneficial fungi interactions are presented and the most common methods to collect and analyze belowground volatiles are evaluated. Furthermore, we suggest a promising method for future research on belowground VOCs.
Project description:Whiteflies (Hemiptera: Aleyrodidae) are sap-feeding global agricultural pests. These piercing-sucking insects have coevolved with intracellular endosymbiotic bacteria that help to supplement their nutrient-poor plant sap diets with essential amino acids and carotenoids. These obligate, primary endosymbionts have been incorporated into specialized organs called bacteriomes where they sometimes coexist with facultative, secondary endosymbionts. All whitefly species harbor the primary endosymbiont Candidatus Portiera aleyrodidarum and have a variable number of secondary endosymbionts. The secondary endosymbiont complement harbored by the cryptic whitefly species Bemisia tabaci is particularly complex with various assemblages of seven different genera identified to date. In this review, we discuss whitefly associated primary and secondary endosymbionts. We focus on those associated with the notorious B. tabaci species complex with emphasis on their biological characteristics and diversity. We also discuss their interactions with phytopathogenic begomoviruses (family Geminiviridae), which are transmitted exclusively by B. tabaci in a persistent-circulative manner. Unraveling the complex interactions of these endosymbionts with their insect hosts and plant viruses could lead to advancements in whitefly and whitefly transmitted virus management.
Project description:The growing concern over the harmful effects of synthetic colorants on both the consumer and the environment has raised a strong interest in natural coloring alternatives. As a result the worldwide demand for colorants of natural origin is rapidly increasing in the food, cosmetic and textile sectors. Natural colorants have the capacity to be used for a variety of industrial applications, for instance, as dyes for textile and non-textile substrates such as leather, paper, within paints and coatings, in cosmetics, and in food additives. Currently, pigments and colorants produced through plants and microbes are the primary source exploited by modern industries. Among the other non-conventional sources, filamentous fungi particularly ascomycetous and basidiomycetous fungi (mushrooms), and lichens (symbiotic association of a fungus with a green alga or cyanobacterium) are known to produce an extraordinary range of colors including several chemical classes of pigments such as melanins, azaphilones, flavins, phenazines, and quinines. This review seeks to emphasize the opportunity afforded by pigments naturally found in fungi as a viable green alternative to current sources. This review presents a comprehensive discussion on the capacity of fungal resources such as endophytes, halophytes, and fungi obtained from a range or sources such as soil, sediments, mangroves, and marine environments. A key driver of the interest in fungi as a source of pigments stems from environmental factors and discussion here will extend on the advancement of greener extraction techniques used for the extraction of intracellular and extracellular pigments. The search for compounds of interest requires a multidisciplinary approach and techniques such as metabolomics, metabolic engineering and biotechnological approaches that have potential to deal with various challenges faced by pigment industry.
Project description:A residue from the primary treatment of a Wastewater Treatment Plant (WWTP) was used to isolate filamentous fungi with lipase production potential. Two of the 27 isolated fungi presented high hydrolysis index and were selected for lipase production by solid-state fermentation (SSF). The fermentations were conducted at 30 °C for 48 h, with moist air circulation, using 20% (w/w) of the residue mixture with a basal medium (agroindustrial residue-babassu cake), obtaining a solid enzymatic preparation (SEP) with lipase activity of 19 U/g with the fungus identified as Aspergillus terreus. Scum, collected in an anaerobic reactor operating in a WWTP, was hydrolyzed with SEP and subjected to anaerobic biodegradability tests at 30 °C. Different dilutions of crude (Control) or hydrolyzed scum in raw sewage were evaluated. The dilution of 5% (v/v) of hydrolyzed scum in raw sewage proved the most adequate, as it resulted in higher methane yield compared to the raw sewage (196 and 133 mL CH4/g CODadded, respectively), without increasing the chemical oxygen demand (COD) of the treated sewage (138 and 134 mg/L). The enzymatic hydrolysis of the scum, followed by dilution in the influent sewage, is technically feasible and increases methane production in anaerobic reactors.
Project description:Reptiles and amphibians (herptiles) are some of the most endangered and threatened species on the planet and numerous conservation strategies are being implemented with the goal of ensuring species recovery. Little is known, however, about the gut microbiome of wild herptiles and how it relates to the health of these populations. Here, we report results from the gut microbiome characterization of both a broad survey of herptiles, and the correlation between the fungus Basidiobolus, and the bacterial community supported by a deeper, more intensive sampling of Plethodon glutinosus, known as slimy salamanders. We demonstrate that bacterial communities sampled from frogs, lizards, and salamanders are structured by the host taxonomy and that Basidiobolus is a common and natural component of these wild gut microbiomes. Intensive sampling of multiple hosts across the ecoregions of Tennessee revealed that geography and host:geography interactions are strong predictors of distinct Basidiobolus operational taxonomic units present within a given host. Co-occurrence analyses of Basidiobolus and bacterial community diversity support a correlation and interaction between Basidiobolus and bacteria, suggesting that Basidiobolus may play a role in structuring the bacterial community. We further the hypothesis that this interaction is advanced by unique specialized metabolism originating from horizontal gene transfer from bacteria to Basidiobolus and demonstrate that Basidiobolus is capable of producing a diversity of specialized metabolites including small cyclic peptides.IMPORTANCEThis work significantly advances our understanding of biodiversity and microbial interactions in herptile microbiomes, the role that fungi play as a structural and functional members of herptile gut microbiomes, and the chemical functions that structure microbiome phenotypes. We also provide an important observational system of how the gut microbiome represents a unique environment that selects for novel metabolic functions through horizontal gene transfer between fungi and bacteria. Such studies are needed to better understand the complexity of gut microbiomes in nature and will inform conservation strategies for threatened species of herpetofauna.