ONE-POT ENZIMATIC DEPOLYMERIZATION OF CELLULOSE IN IONIC LIQUIDS

Green alternatives to fossil-based fuels are very attractive and can be produced from cellulosic materials. Cellulose is the primary product of photosynthesis in plants and has immense importance as a renewable raw material. The production of biofuels starting from cellulose is gaining increasing attention and obviously implies the partial or total hydrolysis of cellulose: enzymatic processes are considered the most promising technology [1]. Cellulases (EC 3.2.1.4) are the enzymes most commonly employed to selectively depolymerize cellulose in buffered aqueous solvents. Because of the very low solubility of cellulose due to its highly organized structure, enzymatic conversions proceed at very slow reaction rates and require the dissolution in a solvent to facilitate the access of cellulases to cellulosic substrates. To improve the yield of fermentable monosaccharides, pretreatments of cellulose, such as thermal, chemical or physical treatment, have been applied to afford a better enzymatic conversion [2]. Ionic liquids (ILs) have been increasingly recognized as excellent solvents for dissolution and pretreatment of cellulose but it was previously reported that ILs induce usually fast enzyme deactivation by protein unfolding [3]. In the present work we present a study on a single-batch, homogeneous phase enzymatic hydrolysis of cellulose using three commercial ILs. We have tested two native proteins from Trichoderma reesei and Humicola insolens and two engineered proteins from T. reesei and Streptomyces sp.. In some cases ILs don’t denature the cellulases used but increase their operational stability as compared to standard buffer solutions and facilitate the dissolution of cellulose. Interestingly, the stability of the four cellulases in the presence of the ILs allows to set-up a procedure lacking of the cellulose pretreatment step. We believe that this strategy could be amenable of scale-up and innovative industrial applications for the efficient one-batch conversion of inexpensive cellulosic materials into derivatives (biofuels, derivatized cellulose, monosaccharides for fine chemicals, etc.) with high potential commercial interest and in the framework of environmentally friendly chemistry. References [1] A.P. dadi, S. Varanasi, C.A. Schall. Biotechnol Bioeng, 95(5), 904-910, (2006). [2] M.B. Turner, S.K. Spear, J.G. Huddleston, J.D. Holbrey, R.D. Rogers. Green Chem, 5(4), 443-447, (2003). [3] S.D. Zhu, Y.X. Wu, Q.M. Chen, C. Wang, S. Jin, Y. Ding, G. Wu, Green Chem, 8, 325-327, (2006)

A METABOLIC-LIKE CYCLE FOR SYNTHETIC APPLICATIONS

Systems Biocatalysis is a new approach consisting of organizing enzymes in vitro to generate an artificial metabolism for synthetic purposes. The interconversion of functional groups is the main objective of biocatalysis, and systems organizing a series of enzymes to achieve a multi-step reaction have been reported. The assembly of essentially the same enzymes utilized in Nature to drive the transformation of carbohydrates towards useful synthetic intermediates [1] has been referred to as an artificial metabolism. SysBiocat aims at a similar goal addressing the generalization and organization of group of enzymes (a tool-box) able to perform a series of reactions of general synthetic utility where the feasibility is connected with the obtainment of enzymes of wide substrate specificity or in a rich array of variable common catalytic functions. [2] As a demonstration of this concept, we have recently assembled a biochemical like cycle (Asp-cycle) connecting among them an unsaturated carboxylate (fumaric acid), an alpha-amino acid (L-aspartic acid), a keto acid (oxalacetic acid) and the corresponding alpha-hydroxyacid (D- or L-malic acid). [3] In this view, the obtained cycle may be exploited by coupling it with synthetically relevant reactions which are driven to completion thanks to one or more irreversible steps in the reaction sequence. ____ [1] W.D. Fessner, C. Walter, “Artificial metabolism”, Angew Chem Int Ed, 1992, 31, p. 614 [2] U. T. Bornscheuer, G. W. Huisman, R. J. Kazlauskas, S. Lutz, J. C. Moore, K. Robins, “Engineering The Third Wave Of Biocatalysis”, Nature, 2012, 485, p. 185 [3] D. Tessaro, L. Pollegioni, L. Piubelli, P. D’Arrigo, S. Servi, “Systems Biocatalysis: An Artificial Metabolism for Interconversion of Functional Groups”, ACS Catalysis, 2015, 5, p. 160

Black Locust (Robinia pseudoacacia L.) Root Cuttings: Diversity and Identity Revealed by SSR Genotyping: A Case Study

Background and Purpose: Black locust (Robinia pseudoacacia L.) is a valuable species native to North America and today widely planted throughout the world for biomass production. In Hungary, where Robinia has great importance in the forest management, the clones have been selected for plantations on good, medium and poor quality sites. To conserve the identity, superior clones are vegetatively propagated by root cuttings. At times the collection of root cuttings can cause uncertainty for clonal identity because of the overlap of roots from neighboring plants. This can occur especially when the repository is damaged from severe environmental accidents and the planting layout has been lost. The aim of this study has been to verify by molecular markers the diversity or identity of black locust clones by root cuttings harvested in a damaged trial. Materials and Methods: Root cuttings of 91 clones belonging to five cultivars were collected in a trial severely damaged by storms and flooding periods. The obtained plantlets were analyzed with nine microsatellite (SSR) markers and the genetic identity/diversity within and among the plants was tested using the software GenAlEx version 6. Results: Multilocus genotypes (MLG) and the Paetkau’s assignation test (1985) revealed genetic variability among the samples: the analyzed plantlets were grouped in four classes instead of the five expected. In addition, 6 unique genotypes have been detected. Conclusions: This study remarks problems that may arise during the harvest of Robinia’s root cuttings, especially when the planting layout has been confused. Molecular analyses can be successfully used to control the germplasm before its sale as guaranty for nurseries, farmers and stakeholders

Rethinking the history of common walnut (Juglans regia L.) in Europe: Its origins and human interactions

Common walnut (Juglans regia L) is an economically important species cultivated worldwide for its high-quality wood and nuts. It is generally accepted that after the last glaciation J. regia survived and grew in almost completely isolated stands in Asia, and that ancient humans dispersed walnuts across Asia and into new habitats via trade and cultural expansion. The history of walnut in Europe is a matter of debate, however. In this study, we estimated the genetic diversity and structure of 91 Eurasian walnut populations using 14 neutral microsatellites. By integrating fossil pollen, cultural, and historical data with population genetics, and approximate Bayesian analysis, we reconstructed the demographic history of walnut and its routes of dispersal across Europe. The genetic data confirmed the presence of walnut in glacial refugia in the Balkans and western Europe. We conclude that human-mediated admixture between Anatolian and Balkan walnut germplasm started in the Early Bronze Age, and between western Europe and the Balkans in eastern Europe during the Roman Empire. A population size expansion and subsequent decline in northeastern and western Europe was detected in the last five centuries. The actual distribution of walnut in Europe resulted from the combined effects of expansion/contraction from multiple refugia after the Last Glacial Maximum and its human exploitation over the last 5,000 years

Ancient humans influenced the current spatial genetic structure of common walnut populations in Asia

Common walnut (Juglans regia L) is an economically important species cultivated worldwide for its wood and nuts. It is generally accepted that J. regia survived and grew spontaneously in almost completely isolated stands in its Asian native range after the Last Glacial Maximum. Despite its natural geographic isolation, J. regia evolved over many centuries under the influence of human management and exploitation. We evaluated the hypothesis that the current distribution of natural genetic resources of common walnut in Asia is, at least in part, the product of ancient anthropogenic dispersal, human cultural interactions, and afforestation. Genetic analysis combined with ethno-linguistic and historical data indicated that ancient trade routes such as the Persian Royal Road and Silk Road enabled long-distance dispersal of J. regia from Iran and Trans-Caucasus to Central Asia, and from Western to Eastern China. Ancient commerce also disrupted the local spatial genetic structure of autochthonous walnut populations between Tashkent and Samarkand (Central- Eastern Uzbekistan), where the northern and central routes of the Northern Silk Road converged. A significant association between ancient language phyla and the genetic structure of walnut populations is reported even after adjustment for geographic distances that could have affected both walnut gene flow and human commerce over the centuries. Beyond the economic importance of common walnut, our study delineates an alternative approach for understanding how the genetic resources of long-lived perennial tree species may be affected by the interaction of geography and human history

Serine metabolism during differentiation of human iPSC-derived astrocytes

: Astrocytes are essential players in development and functions, being particularly relevant as regulators of brain energy metabolism, ionic homeostasis and synaptic transmission. They are also the major source of l-serine in the brain, which is synthesized from the glycolytic intermediate 3-phosphoglycerate through the phosphorylated pathway. l-Serine is the precursor of the two main co-agonists of the N-methyl-d-aspartate receptor, glycine and d-serine. Strikingly, dysfunctions in both l- and d-serine metabolism are associated with neurological and psychiatric disorders. Here, we exploited a differentiation protocol, based on the generation of human mature astrocytes from neural stem cells, and investigated the modification of the proteomic and metabolomic profile during the differentiation process. We show that differentiated astrocytes are more similar to mature rather than to reactive ones, and that axogenesis and pyrimidine metabolism increase up to 30 days along with the folate cycle and sphingolipid metabolism. Consistent with the proliferation and cellular maturation processes that are taking place, also the intracellular levels of l-serine, glycine, threonine, l- and d-aspartate (which level is unexpectedly higher than that of d-serine) show the same biosynthetic time course. A significant utilization of l-serine from the medium is apparent while glycine is first consumed and then released with a peak at 30 days, parallel to its intracellular level. These results underline how metabolism changes during astrocyte differentiation, highlight that d-serine synthesis is restricted in differentiated astrocytes and provide a valuable model for developing potential novel therapeutic approaches to address brain diseases, especially the ones related to serine metabolism alterations

engineering methionine γ lyase from citrobacter freundii for anticancer activity

Abstract Methionine deprivation of cancer cells, which are deficient in methionine biosynthesis, has been envisioned as a therapeutic strategy to reduce cancer cell viability. Methionine γ-lyase (MGL), an enzyme that degrades methionine, has been exploited to selectively remove the amino acid from cancer cell environment. In order to increase MGL catalytic activity, we performed sequence and structure conservation analysis of MGLs from various microorganisms. Whereas most of the residues in the active site and at the dimer interface were found to be conserved, residues located in the C-terminal flexible loop, forming a wall of the active site entry channel, were found to be variable. Therefore, we carried out site-saturation mutagenesis at four independent positions of the C-terminal flexible loop, P357, V358, P360 and A366 of MGL from Citrobacter freundii, generating libraries that were screened for activity. Among the active variants, V358Y exhibits a 1.9-fold increase in the catalytic rate and a 3-fold increase in KM, resulting in a catalytic efficiency similar to wild type MGL. V358Y cytotoxic activity was assessed towards a panel of cancer and nonmalignant cell lines and found to exhibit IC50 lower than the wild type. The comparison of the 3D-structure of V358Y MGL with other MGL available structures indicates that the C-terminal loop is either in an open or closed conformation that does not depend on the amino acid at position 358. Nevertheless, mutations at this position allosterically affects catalysis

How much biodiversity is concealed in the word ‘biodiversity’?

Non peer reviewe

Landscape genetics structure of European sweet chestnut (Castanea sativa Mill): indications for conservation priorities

Sweet chestnut is a tree of great economic (fruit and wood production), ecological and cultural importance in Europe. A large-scale landscape genetic analysis of natural populations of sweet chestnut across Europe is applied to 1) evaluate the geographic patterns of genetic diversity 2) identify spatial coincidences between genetic discontinuities and geographic barriers 3) propose certain chestnut populations as reservoirs of genetic diversity for conservation and breeding programmes. Six polymorphic microsatellite markers were used for genotyping 1608 wild trees sampled in 73 European sites. The Geostatistical IDW technique (ArcGIS 9.3) was used to produce maps of genetic diversity parameters (He, Ar, PAr) and a synthetic map of the population membership (Q value) to the different gene pools. Genetic barriers were investigated using BARRIER 2.2 software and their locations were overlaid on a Digital Elevation Model (GTOPO30). The DIVA-GIS software was used to propose priority areas for conservation. High values of genetic diversity (He) and allelic richness (Ar) were observed in the central area of C. sativa's European distribution range. The highest values of private allelic richness (PAr) were found in the eastern area. Three main gene pools and a significant genetic barrier separating the eastern from the central and western populations were identified. Areas with high priority for genetic conservation were indicated in Georgia, eastern Turkey and Italy. Our results increase knowledge of the biogeographic history of C. sativa in Europe, indicate the geographic location of different gene pools and identify potential priority reservoirs of genetic diversity

Diversity and Source of Airborne Microbial Communities at Differential Polluted Sites of Rome

Biogenic fraction of airborne PM10 which includes bacteria, viruses, fungi and pollens, has been proposed as one of the potential causes of the PM10 toxicity. The present study aimed to provide a comprehensive understanding of the microbial community variations associated to PM10, and their main local sources in the surrounding environment in three urban sites of Rome, characterized by differential pollution rate: green area, residential area and polluted area close to the traffic roads. We combined high-throughput amplicon sequencing of the bacterial 16S rRNA gene and the fungal internal transcribed spacer (ITS) region, with detailed chemical analysis of particulate matter sampled from air, paved road surfaces and leaf surfaces of Quercus ilex. Our results demonstrated that bacterial and fungal airborne communities were characterized by the highest alpha-diversity and grouped separately from epiphytic and road dust communities. The reconstruction of source-sink relationships revealed that the resuspension/deposition of road dust from traffic might contribute to the maximum magnitude of microbial exchanges. The relative abundance of extremotolerant microbes was found to be enhanced in epiphytic communities and was associated to a progressively increase of pollution levels as well as opportunistic human pathogenicity in fungal communities