Cell Factories and Nanotechnologies
The knowledge and experience acquired by our team over the years in cell physiology, biochemistry, molecular biology, genomics, proteomics and fermentation technology has created a sustainable knowledge which allows for an inter-relating of both fundamental and applied research fields. The work developed under the scope of our research lines covers an assortment of topics strategically devoted to R&D projects with potential industrial applications but having as the principal basis biotechnological, molecular genetics and recombinant DNA technology approaches. The strategy is based on a close collaboration with distinguished groups of researchers from both national and international institutions. Emphasis is given to the exploitation of genetic tools for the overproduction of peptides, proteins and other biomolecules as well as for the improvement of microbial strains and, in particular, to the use of bacteria and yeasts as “cell factories” for the production of value-added bioproducts.
With the aim of optimising production, distinct bacterial and yeast expression systems have been explored and various methodologies for protein purification have been developed. This has involved: investigation of a variety of expression systems, utilisation of alternative hosts, manipulation of the fermentation conditions by surface response methodology and finally, downstream processing. The sustainability of a bioprocess devoted to the production of a value-added recombinant protein is dictated not only by the simplification of the production and downstream processes, but, and in particular for biomedical applications, also by the reduction of endotoxins to negligible levels.
The projects developed over previous years have focused on: i) production and characterization of recombinant peptides and proteins for biomedical applications; ii) design of bioinspired protein-based performance materials; iii) metabolic engineering and structural modeling of enzymes for the optimisation of industrial processes.
Ongoing and future Work
Design, biological production and characterization of recombinant elastin-like and silk-elastin-like protein polymers – a new class of nano-biomaterials
This very promising and exciting area of research aims at designing, producing and characterising a new class of bioinspired high performance materials: the protein-based polymers (PBPs). The properties of PBPs are attributed to the presence of short repeating amino acid sequences contained in fibrous proteins such as fibroin (GAGAGS) and elastin (VPGVG). By using molecular genetics tools we have produced a set of PBPs based on the amino acid sequence of elastin (Elastin-Like Polymers – ELPs) which are capable of self-assembling into nanoparticles in response to a thermal stimulus. Additionally, by combining the flexible elastin-like blocks with crystalline silk-like blocks we have also produced a new set of protein-based copolymers (Silk-Elastin-Like-Polymers – SELPs), the characteristics and mechanical properties of which are currently under evaluation. The stability of these PBPs in combination with their biocompatibility and unique mechanical characteristics provides the basis for their exploitation in novel biomedical applications. This project is funded by FP7.
Protein engineered hybrid biomaterials for biomedical applications: PBPs fused with bioactive peptides/proteins
This project aims at the development of novel protein-based materials for therapeutic applications. The construction of hybrid biomaterials interrelates with the previous project by creating chimeric genetic fusions between a biomaterial (the structural domain) and a bioactive molecule (the functional domain).
The main goal is to develop novel biomaterials for protein delivery systems as well as to develop new strategies for the functionalisation of materials to be used in various biomedical applications, namely for the production of recombinant Human Bone Morphogenetic Protein-2 (BMP-2) for tissue engineering applications. Fusion of BMP-2 with a thermoresponsive elastin-like tag allows for its purification under mild conditions and for in loco purification and encapsulation of BMP-2 in elastin-like nanoparticles. The production of recombinant antimicrobial peptides for biofunctionalisation of materials and surfaces is another application under development.
Exploring the biotechnological potential of Pseudomonas using high-throughput technologies
Bacteria belonging to the Pseudomonas genus possess an intricate ability to tolerate and thrive in challenging environments such as the human lungs, the deep sea and in highly polluted soils. Such competence resides in their wide nutritional and metabolic versatility and in a complex network of adaptation mechanisms. In other words, the necessary code to efficiently cope with a wide variety of diverse substrates and biotic and/or abiotic stress conditions is included in their genomic catalog. Therefore, they hold great potential as a source of novel biomolecules and cell factories for various biotechnological applications, namely in the fields of biocatalysis, biosensors, bioremediation and biomedicine. The main goal of this topic is to develop the necessary knowledge base and analytical resources to establish selected Pseudomonas spp. and/or their biomolecules as biotechnological tools using an integrative biological approach by the application of high-throughput technologies.
Monoterpene biotranformation in Pseudomonas sp. M1
The M1 strain is able to biotransform a variety of monoterpene compounds. Many of these compounds exhibit properties such as volatility, flavour/aroma and toxicity and play important roles in plant defence, plant-to-plant communication and pollinator attraction. In addition, some monoterpenes have medicinal properties; they may have anti-carcinogenic, analgesic, antimalarial, anti-ulcer, hepaticidal, antimicrobial, antimutagenic and/or diuretic activity. Therefore, there is great interest in fully understanding the molecular mechanistics employed by strain M1 in recognising and biotransforming monoterpenes. Such knowledge may allow for the production of monoterpene derivatives with important biotechnological value. To accomplish such a laborious task, we have sequenced the full genome of M1 and partially characterized its proteome. In addition, transcriptomic and metabolomic strategies are being setup to provide a comprehensive perspective of the monoterpene-associated biomolecule repertoire of this strain. This project is funded by the FCT.
Comparative genomics and immunoproteomics of Pseudomonas aeruginosa clinical isolates
P. aeruginosa, a ubiquitous pathogen found widespread throughout the environment, is a leading cause of opportunistic infections in humans. Presently it is associated with an ever-widening spectrum of infections, most of which are acute and include: bacteraemia, pneumonia, urosepsis and wound infection. The mortality attributable to P. aeruginosa infections such as ventilator-associated pneumonia or bacteraemia is substantial, especially for patients who receive inadequate empirical therapy. In fact many P. aeruginosa isolates often possess a rather high tolerance to antibiotics, with 30-40% of the isolates being resistant to various classes of antibiotics. Our collaboration with Hospital de Braga in Braga, Portugal (where over 700 P. aeruginosa isolates are analysed per year) aims at stimulating a holistic research approach so as to provide relevant information and tools to clinicians to circumvent the multi-resistance phenomena of P. aeruginosa. The main research lines in this project are: i) identification of pathogenicity islands among the clinical isolates via comparative genomics; ii) identification of SNPs among the clinical isolates via comparative genomics; iii) comparison of the immunoproteomes of the clinical isolates using antisera obtained from various P. aeruginosa crude extracts. The integration of the information from these strategies may lead to the identification of P. aeruginosa biomarkers associated with the phenotypic variation that is currently registered for the different clinical isolates, leading to a more efficient diagnostic capacity.
Metabolic engineering of yeast for fuel molecule production
Green fuel ethanol from lignocellulosic hydrolysates is currently a very hot research topic. Efficient metabolism of the sugars L-arabinose and D-xylose is of importance as they make up the majority of the pentose sugars in biomass. We have cloned a putative L-arabinose transporter gene, AmLAT1, from the efficient arabinose fermenter Candida arabinofermentans. We are currently characterising the transporter and investigating its use in improving an industrial yeast strain also expressing metabolic pathways for D-arabinose and D-xylose metabolism. This project has been funded by FP6.
Cold adapted enzymes: fundamentals and applications
Cold adapted enzymes produced by organisms inhabiting permanently low temperature environments have been found to have successfully adapted to their habitat and to retain high catalytic activities even at temperatures close to 0º C. They have attracted attention both from a fundamental (understanding the molecular basis of their adaptation) and applied (their use in low to moderate temperature biotechnological applications) point of view. We are currently focusing on the role of protein dynamics in protein adaptation to different temperatures, using a cold adapted thiol disulphide oxidoreductase as a model enzyme and comparing its structure and dynamics as well as its physico-chemical and biophysical properties with homologous enzymes from mesophilic and thermophilic organisms. We are also currently investigating the use of these enzymes as tools in low to moderate temperature biotechnological applications (e.g food and feed industries) as their high activity as compared to mesophilic and thermophilic enzymes at the temperatures used in these processes, as well as their ease of inactivation indicates a high potential for application.
Bessa, P. C., Machado, R., Nurnberger, S., Dopler, D., Banerjee, A., Cunha, A. M., Rodriguez-Cabello, J. C., Redl, H., van Griensven, M., Reis, R. L., Casal, M. 2010. Thermoresponsive self-assembled elastin-based nanoparticles for delivery of BMPs. J Control Release, 142, 312-318.
Machado, R., Ribeiro, A. J., Padrao, J., Silva, D., Nobre, A., Teixeira, J. A., Arias, F. J., Cunha, A. M., Rodriguez-Cabello, J. C., Casal, M. 2009. Exploiting the sequence of naturally occurring elastin: Construction, production and characterization of a recombinant thermoplastic protein-based polymer. J Nano Res, 6, 133-145.
Araújo, R., Silva, C., Machado, R., Casal, M., Cunha, A. M., Rodriguez-Cabello, J. C., Cavaco-Paulo, A. 2009. Proteolytic enzyme engineering: a tool for wool. Biomacromolecules, 10, 1655-1661.
Santos, P.M., Sá-Correia I. 2009. Adaptation to beta-myrcene catabolism in Pseudomonas sp. M1: an expression proteomics analysis. Proteomics, 9, 5101-5111.
Santos, P.M., Simões, T., Sá-Correia, I. 2009. Insights into yeast adaptive response to the agricultural fungicide mancozeb: a toxicoproteomics approach. Proteomics, 9, 657-670.
Cosme, A.M., Becker, A., Santos, M.R., Sharypova, L.A., Santos, P.M., Moreira, L.M. 2008. The outer membrane protein TolC from Sinorhizobium meliloti affects protein secretion, polysaccharide biosynthesis, antimicrobial resistance and symbiosis. Molecular Plant-Microbe Interactions, 21, 947-957.
Santos, P.M., Roma, V., Benndorf, D., von Bergen, M., Harms, H., Sá-Correia, I. 2007. Mechanistic insights into the global response to phenol in the phenol-biodegrading strain Pseudomonas sp. M1, revealed by quantitative proteomics. OMICS: A Journal of Integrative Biology, 11, 233-251.
Artur Cavaco-Paulo, Margarida Casal, Rita Araújo, Carla Manuela Silva, Process for the treatment of animal fibres aimed at improving the shrink-resistance thereof. International Patent Number WO2010/001356