Amina Antonacci

Optical biosensors for environmental monitoring based on computational and biotechnological tools for engineering the photosynthetic D1 protein of Chlamydomonas reinhardtii

Homology-based protein modelling and computational screening followed by virtual mutagenesis analyses were used to identify functional amino acids in the D1 protein of the photosynthetic electron transfer chain interacting with herbicides. A library of functional mutations in the unicellular green alga Chlamydomonas reinhardtii for preparing biomediators was built and their interactions with herbicides were calculated. D1 proteins giving the lowest and highest binding energy with herbicides were considered as suitable for preparing the environmental biosensors for detecting specific herbicide classes. Arising from the results of theoretical calculations, three mutants were prepared by site-directed mutagenesis and characterized by fluorescence analysis. Their adsorption and selective recognition ability were studied by an equilibrium-adsorption method. The S268C and S264K biomediators showed high sensitivity and resistance, respectively, to both triazine and urea classes of herbicides. When immobilized on a silicon septum, the biomediators were found to be highly stable, remaining so for at least 1-month at room temperature. The fluorescence properties were exploited and a reusable and portable multiarray optical biosensor for environmental monitoring was developed with limits of detection between 0.8 x 10(-11) and 3.0 x 10(-9), depending on the target analyte. In addition, biomediator regeneration without obvious deterioration in performance was demonstrated.

Structure‐based design of novel Chlamydomonas reinhardtii D1‐D2 photosynthetic proteins for herbicide monitoring

The D1‐D2 heterodimer in the reaction center core of phototrophs binds the redox plastoquinone cofactors, QA and QB, the terminal acceptors of the photosynthetic electron transfer chain in the photosystem II (PSII). This complex is the target of the herbicide atrazine, an environmental pollutant competitive inhibitor of QB binding, and consequently it represents an excellent biomediator to develop biosensors for pollutant monitoring in ecosystems. In this context, we have undertaken a study of the Chlamydomonas reinhardtii D1‐D2 proteins aimed at designing site directed mutants with increased affinity for atrazine. The three‐dimensional structure of the D1 and D2 proteins from C. reinhardtii has been homology modeled using the crystal structure of the highly homologous Thermosynechococcus elongatus proteins as templates. Mutants of D1 and D2 were then generated in silico and the atrazine binding affinity of the mutant proteins has been calculated to predict mutations able to increase PSII affinity for atrazine. The computational approach has been validated through comparison with available experimental data and production and characterization of one of the predicted mutants. The latter analyses indicated an increase of one order of magnitude of the mutant sensitivity and affinity for atrazine as compared to the control strain. Finally, D1‐D2 heterodimer mutants were designed and selected which, according to our model, increase atrazine binding affinity by up to 20 kcal/mol, representing useful starting points for the development of high affinity biosensors for atrazine.

Structure/function/dynamics of photosystem II plastoquinone binding sites.

Photosystem II (PSII) continuously attracts the attention of researchers aiming to unravel the riddle of its functioning and efficiency fundamental for all life on Earth. Besides, an increasing number of biotechnological applications have been envisaged exploiting and mimicking the unique properties of this macromolecular pigment-protein complex. The PSII organization and working principles have inspired the design of electrochemical water splitting schemes and charge separating triads in energy storage systems as well as biochips and sensors for environmental, agricultural and industrial screening of toxic compounds. An intriguing opportunity is the development of sensor devices, exploiting native or manipulated PSII complexes or ad hoc synthesized polypeptides mimicking the PSII reaction centre proteins as bio-sensing elements. This review offers a concise overview of the recent improvements in the understanding of structure and function of PSII donor side, with focus on the interactions of the plastoquinone cofactors with the surrounding environment and operational features. Furthermore, studies focused on photosynthetic proteins structure/function/dynamics and computational analyses aimed at rational design of high-quality bio-recognition elements in biosensor devices are discussed.

Healthy and adverse effects of plant-derived functional metabolites: the need of revealing their content and bioactivity in a complex food matrix.

In recent years, both food quality and its effect on human health have become a fundamental issue all over the world. As a consequence of this new and increased awareness, American, European, and Asian policymakers have strongly encouraged the research programs on food quality and safety thematic. Attempts to improve human health and to satisfy peoples desire for healthcare without intake of pharmaceuticals, has led the food industry to focus attention on functional or nutraceutical food. For a long time, compounds with nutraceutical activity have been produced chemically, but the new demands for a sustainable life have gradually led the food industry to move towards natural compounds, mainly those derived from plants. Many phytochemicals are known to promote good health, but, sometimes, undesirable effects are also reported. Furthermore, several products present on the market show few benefits and sometimes even the reverse - unhealthy effects; the evidence of efficacy is often unconvincing and epidemiological studies are necessary to prove the truth of their claims. Therefore, there is a need for reliable analytical control systems to measure the bioactivity, content, and quality of these additives in the complex food matrix. This review describes the most widespread nutraceutics and an analytical control of the same using recently developed biosensors which are promising candidates for routine control of functional foods.

Photosynthesis at the forefront of a sustainable life.

The development of a sustainable bio-based economy has drawn much attention in recent years, and research to find smart solutions to the many inherent challenges has intensified. In nature, perhaps the best example of an authentic sustainable system is oxygenic photosynthesis. The biochemistry of this intricate process is empowered by solar radiation influx and performed by hierarchically organized complexes composed by photoreceptors, inorganic catalysts, and enzymes which define specific niches for optimizing light-to-energy conversion. The success of this process relies on its capability to exploit the almost inexhaustible reservoirs of sunlight, water, and carbon dioxide to transform photonic energy into chemical energy such as stored in adenosine triphosphate. Oxygenic photosynthesis is responsible for most of the oxygen, fossil fuels, and biomass on our planet. So, even after a few billion years of evolution, this process unceasingly supports life on earth, and probably soon also in outer-space, and inspires the development of enabling technologies for a sustainable global economy and ecosystem. The following review covers some of the major milestones reached in photosynthesis research, each reflecting lasting routes of innovation in agriculture, environmental protection, and clean energy production.

Directed evolution and in silico analysis of reaction centre proteins reveal molecular signatures of photosynthesis adaptation to radiation pressure.

Evolutionary mechanisms adopted by the photosynthetic apparatus to modifications in the Earths atmosphere on a geological time-scale remain a focus of intense research. The photosynthetic machinery has had to cope with continuously changing environmental conditions and particularly with the complex ionizing radiation emitted by solar flares. The photosynthetic D1 protein, being the site of electron tunneling-mediated charge separation and solar energy transduction, is a hot spot for the generation of radiation-induced radical injuries. We explored the possibility to produce D1 variants tolerant to ionizing radiation in Chlamydomonas reinhardtii and clarified the effect of radiation-induced oxidative damage on the photosynthetic proteins evolution. In vitro directed evolution strategies targeted at the D1 protein were adopted to create libraries of chlamydomonas random mutants, subsequently selected by exposures to radical-generating proton or neutron sources. The common trend observed in the D1 aminoacidic substitutions was the replacement of less polar by more polar amino acids. The applied selection pressure forced replacement of residues more sensitive to oxidative damage with less sensitive ones, suggesting that ionizing radiation may have been one of the driving forces in the evolution of the eukaryotic photosynthetic apparatus. A set of the identified aminoacidic substitutions, close to the secondary plastoquinone binding niche and oxygen evolving complex, were introduced by site-directed mutagenesis in un-transformed strains, and their sensitivity to free radicals attack analyzed. Mutants displayed reduced electron transport efficiency in physiological conditions, and increased photosynthetic performance stability and oxygen evolution capacity in stressful high-light conditions. Finally, comparative in silico analyses of D1 aminoacidic sequences of organisms differently located in the evolution chain, revealed a higher ratio of residues more sensitive to oxidative damage in the eukaryotic/cyanobacterial proteins compared to their bacterial orthologs. These results led us to hypothesize an archaean atmosphere less challenging in terms of ionizing radiation than the present one.

A powerful molecular engineering tool provided efficient Chlamydomonas mutants as bio-sensing elements for herbicides detection.

This study was prompted by increasing concerns about ecological damage and human health threats derived by persistent contamination of water and soil with herbicides, and emerging of bio-sensing technology as powerful, fast and efficient tool for the identification of such hazards. This work is aimed at overcoming principal limitations negatively affecting the whole-cell-based biosensors performance due to inadequate stability and sensitivity of the bio-recognition element. The novel bio-sensing elements for the detection of herbicides were generated exploiting the power of molecular engineering in order to improve the performance of photosynthetic complexes. The new phenotypes were produced by an in vitro directed evolution strategy targeted at the photosystem II (PSII) D1 protein of Chlamydomonas reinhardtii, using exposures to radical-generating ionizing radiation as selection pressure. These tools proved successful to identify D1 mutations conferring enhanced stability, tolerance to free-radical-associated stress and competence for herbicide perception. Long-term stability tests of PSII performance revealed the mutants capability to deal with oxidative stress-related conditions. Furthermore, dose-response experiments indicated the strains having increased sensitivity or resistance to triazine and urea type herbicides with I50 values ranging from 6×10−8 M to 2×10−6 M. Besides stressing the relevance of several amino acids for PSII photochemistry and herbicide sensing, the possibility to improve the specificity of whole-cell-based biosensors, via coupling herbicide-sensitive with herbicide-resistant strains, was verified.

Mutations of Photosystem II D1 Protein That Empower Efficient Phenotypes of Chlamydomonas reinhardtii under Extreme Environment in Space

Space missions have enabled testing how microorganisms, animals and plants respond to extra-terrestrial, complex and hazardous environment in space. Photosynthetic organisms are thought to be relatively more prone to microgravity, weak magnetic field and cosmic radiation because oxygenic photosynthesis is intimately associated with capture and conversion of light energy into chemical energy, a process that has adapted to relatively less complex and contained environment on Earth. To study the direct effect of the space environment on the fundamental process of photosynthesis, we sent into low Earth orbit space engineered and mutated strains of the unicellular green alga, Chlamydomonas reinhardtii, which has been widely used as a model of photosynthetic organisms. The algal mutants contained specific amino acid substitutions in the functionally important regions of the pivotal Photosystem II (PSII) reaction centre D1 protein near the QB binding pocket and in the environment surrounding Tyr-161 (YZ) electron acceptor of the oxygen-evolving complex. Using real-time measurements of PSII photochemistry, here we show that during the space flight while the control strain and two D1 mutants (A250L and V160A) were inefficient in carrying out PSII activity, two other D1 mutants, I163N and A251C, performed efficient photosynthesis, and actively re-grew upon return to Earth. Mimicking the neutron irradiation component of cosmic rays on Earth yielded similar results. Experiments with I163N and A251C D1 mutants performed on ground showed that they are better able to modulate PSII excitation pressure and have higher capacity to reoxidize the QA − state of the primary electron acceptor. These results highlight the contribution of D1 conformation in relation to photosynthesis and oxygen production in space.

The NUTRA-SNACKS Project: Basic Research and Biotechnological Programs on Nutraceutics

The Nutra-Snacks project aims at creating novel high quality ready-to-eat foods with functional activity, useful for promoting public health. The team is composed of seven research institutes and three SMEs from different countries whose activities span from basic to applied research providing the right technological transfer to small and medium industries involved in the novel food production chain. Strategic objectives include the application of plant cell and in vitro culture systems to create very large amounts of high-value plant secondary metabolites with recognized anticancer, antilipidemic, anticholesterol, antimicrobial, antiviral, antihypertensive and anti-inflammatory properties and to include them in specific food products. To this end, the screening of a vast number of working organisms capable of accumulating the desired compounds and the characterization of their expression profiles represent fundamental steps in the research program. The information allows the identification of plant species hyper-producing metabolites and selection of those metabolites capable of specifically counteracting the oxidative stress that underlies the development of important pathologies and diseases. In addition, devising safe metabolite extraction procedures is also crucial in order to provide nutraceutical-enriched extracts compatible with human health. New biotechnological approaches are also undertaken including the exploitation of photosynthetic algal strains in bio-farms to enhance the synthesis ofantioxidant compounds and the design of novel bioreactors for small and large scale biomass production. Further outstanding objectives include the development of (i) safety and quality control protocols (ii) biosensor techniques for the analysis of the emerging ready-to-eat food and (iii) a contribution to define a standard for new regulations on nutraceutics.

Computational Biology, Protein Engineering, and Biosensor Technology: a Close Cooperation for Herbicides Monitoring

Application of herbicides has led to a marked increase in the productivity and preservation of agricultural products, as a result of which, cultural techniques for weed control, such as altering soil pH, salinity, fertility levels or mechanical approaches, have been abandoned. These compounds are also used extensively in industrial sites, roadsides, ditch banks, irrigation canals, fence lines, recreational areas, lawns, railroad embankments, and power line rights-of-way, to remove undesirable plants that might cause damage, present fire hazards, or impede work crews. They also reduce costs of mowing procedures. However, due to the toxic effect, their control is carried out by a system of national registration which limits the manufacture and/or sale of pesticide products to those who have been approved (Montesinos 2003). In this context, herbicides were classified into families based on their chemical similarity or, as proposed by the global Herbicide Resistance Action Committee (HRAC) group, according to their target sites and modes of action (Table 1). Standards and regulations for the classifications, labelling, and packaging of pesticides were first set up by the EUROPEAN ECONOMIC COMMUNITY (EEC) Council Directive 67/548/ in 1967. At present the issue regarding herbicides is quite intricate, because according to the Food and Agriculture Organization (FAO), their exclusion would lead to a strong reduction in farming production; however, several toxic effects on biological systems associated with their use were proved by epidemiological and experimental studies (Waller et al., 2010; Roberts et al., 2010; Frazier 2007). After the first cases of animals poisoned by heavy utilization of herbicides, the monitoring of these compounds to avoid accumulation in the human body were strongly intensified. In particular in 1963, the World Health Organization (WHO) and FAO created the Codex Alimentarius Commission, which joined 173 signatories from the European Community (EC) countries in order to control the tolerable limits of pollutants in food. Twenty years later, the EC established a legal framework for the regulation of pesticides in all member countries. The Commission is responsible for the registration of pesticides actively used in all European countries. This authority is granted