Eunice Ferreira

Ferreira, E.A., Pacheco, C.C., Pinto, F., Pereira, J., Lamosa, L., Oliveira, P., Kirov, B., Jaramillo, A., Tamagnini, P. 2018
Cyanobacteria are promising ‘low-cost’ cell factories since they have minimal nutritional requirements, high metabolic plasticity and can use sunlight and CO2 as energy and carbon sources. The unicellular Synechocystis sp. PCC 6803, already considered the ‘green’ Escherichia coli, is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well-characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the Standard European Vector Architecture (SEVA) repository to transform Synechocystis. Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered Synechocystis growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to the reference PrnpB, three of which could be efficiently repressed. As a proof-of-concept, from the expanded toolbox, one promoter was selected and assembled with the ggpS gene [encoding one of the proteins involved in the synthesis of the native compatible solute glucosylglycerol (GG)] and the synthetic device was introduced into Synechocystis using one of the SEVA plasmids. The presence of this device restored the production of the GG in a ggpS deficient mutant validating the functionality of the tools/device developed in this study.

Lindblad, P., Fuente, D., Borbe, F., Cicchi, B., Conejero, J.A., Couto, N., Čelešnik, H., Diano, M.M., Dolinar, M., Esposito, S., Evans, C., Ferreira, E.A., et al., 2019
CyanoFactory, Design, construction and demonstration of solar biofuel production using novel (photo)synthetic cell factories, was an R&D project developed in response to the European Commission FP7-ENERGY-2012-1 call “Future Emerging Technologies” and the need for significant advances in both new science and technologies to convert solar energy into a fuel. CyanoFactory was an example of “purpose driven” research and development with identified scientific goals and creation of new technologies. The present overview highlights significant outcomes of the project, three years after its successful completion.
The scientific progress of CyanoFactory involved: (i) development of a ToolBox for cyanobacterial synthetic biology; (ii) construction of DataWarehouse/Bioinformatics web-based capacities and functions; (iii) improvement of chassis growth, functionality and robustness; (iv) introduction of custom designed genetic constructs into cyanobacteria, (v) improvement of photosynthetic efficiency towards hydrogen production; (vi) biosafety mechanisms; (vii) analyses of the designed cyanobacterial cells to identify bottlenecks with suggestions on further improvements; (viii) metabolic modelling of engineered cells; (ix) development of an efficient laboratory scale photobioreactor unit; and (x) the assembly and experimental performance assessment of a larger (1350 L) outdoor flat panel photobioreactor system during two seasons.
CyanoFactory - Custom design and purpose construction of microbial cells for the production of desired products using synthetic biology – aimed to go beyond conventional paths to pursue innovative and high impact goals. CyanoFactory brought together ten leading European partners (universities, research organizations and enterprises) with a common goal – to develop the future technologies in Synthetic biology and Advanced photobioreactors.

Kobayashi, S., Nakajima, M., Asano, R., Ferreira, E.A., Abe, K., Tamagnini, P., Atsumi, S., Sode, K. 2019
The development of a versatile tool for the gene regulation in cyanobacteria is critical for the future realization of cyanobacterial bioprocessing. The use of chemical inducers to regulate gene expression are not practical considering their cost and technical difficulty in removing them from culture. Therefore, we have focused on a cyanobacteria-derived chromatic acclimation sensor, the green-light sensing system, CcaS/CcaR two-component system, derived from Synechocystis sp. PCC6803 (PCC6803) as a toll for genetic regulation. However, the regulation of gene expression levels by CcaS is not strict. We have previously developed a miniaturized CcaS, CcaS#11, which is a truncated CcaS showing gene induction under red-light illumination and strict repression under green-light illumination in Escherichia coli. In this study, CcaS#11 was transformed in cyanobacteria to achieve red-light-regulated gene expression in cyanobacteria. The application was first attempted in PCC6803 after knocking out genomic CcaS/CcaR system to exclude interference. The results revealed gene expression was only induced under red-light illumination and strictly repressed under green-light illumination. The red-light-regulated gene expression was also applied for a marine cyanobacteria, Synechococcus sp. NKBG15041c (NKBG15041c). In NKBG15041c, gene expression was induced under red-light illumination and strictly repressed under green-light illumination with a 2-fold higher ON/OFF ratio compared with the original CcaS/CcaR two-component system. Therefore, the constructed red-light-regulated gene expression system using CcaS#11 has a great potential as a platform technology for the further development of light-regulated bioprocesses with strict control in cyanobacteria.

Campos, J., Moreira, C., Costa-Dias, S., Ferreira, E., Matos, A., Vasconcelos, V., Antunes, A. 2020
Studying the diet of crustaceans based on the direct visual inspection of the gut contents can be difficult because they can ingest only a portion of the prey and further crush it into very small pieces, making a very few food items identifiable. Here, the usefulness of molecular methods to identify the species present in the gut contents of two crustaceans, the shore crab Carcinus maenas and the brown shrimp Crangon crangon, was tested. Stomach contents were removed, and their total genomic DNA extracted followed by amplification (PCR) with four specific prey mtDNA primers. Since both crustaceans are opportunistic predators, preying upon around 40 different species, the study was restricted to common potential preys in the sampled area, the Minho Estuary (Portugal): the common goby Pomatoschistus microps, the ragworm Hediste diversicolor, the flounder Platichthys flesus and the peppery furrow shell Scrobicularia plana. After testing the most appropriate methodology for DNA extraction and PCR amplification to detect the presence of each potential prey with positive control samples, the same methodology was applied to the stomach content samples. The molecular methods allowed for detection of the presence of P. microps, P. flesus and H. diversicolor in the stomachs of both crustaceans. Therefore, once potential prey and their availability in the system are known, PCR technique can be an alternative tool to overcome some of the limitations of traditional methods in the study of the crustaceans’ diet, even when stomach contents include a great part of ‘detritus’. This study reflects also the need to develop more tools (primers) to improve the assessment of predator–prey interactions in aquatic ecosytems.