Assessing the Antitumor Potential of Variants of the Extracellular Carbohydrate Polymer from Synechocystis ΔsigF Mutant.

Cancer is a leading cause of death worldwide with a huge societal and economic impact. Clinically effective and less expensive anticancer agents derived from natural sources can help to overcome limitations and negative side effects of chemotherapy and radiotherapy. Previously, we showed that the extracellular carbohydrate polymer of a Synechocystis ΔsigF overproducing mutant displayed a strong antitumor activity towards several human tumor cell lines, by inducing high levels of apoptosis through p53 and caspase-3 activation. Here, the ΔsigF polymer was manipulated to obtain variants that were tested in a human melanoma (Mewo) cell line. Our results demonstrated that high molecular mass fractions were important for the polymer bioactivity, and that the reduction of the peptide content generated a variant with enhanced in vitro antitumor activity. This variant, and the original ΔsigF polymer, were further tested in vivo using the chick chorioallantoic membrane (CAM) assay. Both polymers significantly decreased xenografted CAM tumor growth and affected tumor morphology, by promoting less compact tumors, validating their antitumor potential in vivo. This work contributes with strategies for the design and testing tailored cyanobacterial extracellular polymers and further strengths the relevance of evaluating this type of polymers for biotechnological/biomedical applications.

Towards the rate limit of heterologous biotechnological reactions in recombinant cyanobacteria.

BACKGROUND: Cyanobacteria have emerged as highly efficient organisms for the production of chemicals and biofuels. Yet, the productivity of the cell has been low for commercial application. Cyanobacterial photobiotransformations utilize photosynthetic electrons to form reducing equivalents, such as NADPH-to-fuel biocatalytic reactions. These photobiotransformations are a measure to which extent photosynthetic electrons can be deviated toward heterologous biotechnological processes, such as the production of biofuels. By expressing oxidoreductases, such as YqjM from Bacillus subtilis in Synechocystis sp. PCC 6803, a high specific activity was obtained in the reduction of maleimides. Here, we investigated the possibility to accelerate the NAD(P)H-consuming redox reactions by addition of carbohydrates as exogenous carbon sources such as D-Glucose under light and darkness. RESULTS: A 1.7-fold increase of activity (150 µmol min-1 gDCW-1) was observed upon addition of D-Glucose at an OD750 = 2.5 (DCW = 0.6 g L-1) in the biotransformation of 2-methylmaleimide. The stimulating effect of D-Glucose was also observed at higher cell densities in light and dark conditions as well as in the reduction of other substrates. No increase in both effective photosynthetic yields of Photosystem II and Photosystem I was found upon D-Glucose addition. However, we observed higher NAD(P)H fluorescence when D-Glucose was supplemented, suggesting increased glycolytic activity. Moreover, the system was scaled-up (working volume of 200 mL) in an internally illuminated Bubble Column Reactor exhibiting a 2.4-fold increase of specific activity under light-limited conditions. CONCLUSIONS: Results show that under photoautotrophic conditions at a specific activity of 90 µmol min-1 gDCW-1, the ene-reductase YqjM in Synechocystis sp. PCC 6803 is not NAD(P)H saturated, which is an indicator that an increase of the rates of heterologous electron consuming processes for catalysis and biofuel production will require funnelling further reducing power from the photosynthetic chain toward heterologous processes.

Subcellular compartmentalization of aluminum reduced its hazardous impact on rye photosynthesis.

Aluminum (Al) toxicity limits crops growth and production in acidic soils. Compared to roots, less is known about the toxic effects of Al in leaves. Al subcellular compartmentalization is also largely unknown. Using rye (Secale cereale L.) Beira (more tolerant) and RioDeva (more sensitive to Al) genotypes, we evaluated the patterns of Al accumulation in leaf cell organelles and the photosynthetic and metabolic changes to cope with Al toxicity. The tolerant genotype accumulated less Al in all organelles, except the vacuoles. This suggests that Al compartmentalization plays a role in Al tolerance of Beira genotype. PSII efficiency, stomatal conductance, pigment biosynthesis, and photosynthesis metabolism were less affected in the tolerant genotype. In the Calvin cycle, carboxylation was compromised by Al exposure in the tolerant genotype. Other Calvin cycle-related enzymes, phoshoglycerate kinase (PGK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), triose-phosphate isomerase (TPI), and fructose 1,6-bisphosphatase (FBPase) activities decreased in the sensitive line after 48 h of Al exposure. Consequentially, carbohydrate and organic acid metabolism were affected in a genotype-specific manner, where sugar levels increased only in the tolerant genotype. In conclusion, Al transport to the leaf and compartmentalization in the vacuoles tolerant genotype's leaf cells provide complementary mechanisms of Al tolerance, protecting the photosynthetic apparatus and thereby sustaining growth.

CRISPRi as a Tool to Repress Multiple Copies of Extracellular Polymeric Substances (EPS)-Related Genes in the Cyanobacterium Synechocystis sp. PCC 6803.

The use of the versatile cyanobacterial extracellular polymeric substances (EPS) for biotechnological/biomedical applications implies an extensive knowledge of their biosynthetic pathways to improve/control polymer production yields and characteristics. The multiple copies of EPS-related genes, scattered throughout cyanobacterial genomes, adds another layer of complexity, making these studies challenging and time-consuming. Usually, this issue would be tackled by generating deletion mutants, a process that in cyanobacteria is also hindered by the polyploidy. Thus, the use of the CRISPRi multiplex system constitutes an efficient approach to addressing this redundancy. Here, three putative Synechocystis sp. PCC 6803 kpsM homologues (slr0977, slr2107, and sll0574) were repressed using this methodology. The characterization of the 3-sgRNA mutant in terms of fitness/growth and total carbohydrates, released and capsular polysaccharides, and its comparison with previously generated single knockout mutants pointed towards Slr0977 being the key KpsM player in Synechocystis EPS production. This work validates CRISPRi as a powerful tool to unravel cyanobacterial complex EPS biosynthetic pathways expediting this type of studies.

The Extremophile Endolithella mcmurdoensis gen. et sp. nov. (Trebouxiophyceae, Chlorellaceae), A New Chlorella-like Endolithic Alga From Antarctica.

The McMurdo Dry Valleys constitute the largest ice-free region of Antarctica and one of the most extreme deserts on Earth. Despite the low temperatures, dry and poor soils and katabatic winds, some microbes are able to take advantage of endolithic microenvironments, inhabiting the pore spaces of soil and constituting photosynthesis-based communities. We isolated a green microalga, Endolithella mcmurdoensis gen. et sp. nov, from an endolithic sandstone sample collected in the McMurdo Dry Valleys (Victoria Land, East Antarctica) during the K020 expedition, in January 2013. The single non-axenic isolate (E. mcmurdoensis LEGE Z-009) exhibits cup-shaped chloroplasts, electron-dense bodies, and polyphosphate granules but our analysis did not reveal any diagnostic morphological characters. On the basis of phylogenetic analysis of the 18S rRNA (SSU) gene, the isolate was found to represent a new genus within the family Chlorellaceae.

Characterization and antitumor activity of the extracellular carbohydrate polymer from the cyanobacterium Synechocystis ΔsigF mutant.

Cyanobacterial extracellular carbohydrate polymers are particularly attractive for biotechnological applications. Previously, we determined the monosaccharidic composition of the polymer of a Synechocystis ΔsigF overproducing mutant. Here, we further characterized this polymer, demonstrated that it is possible to recover it in high yields, and successfully use it for biomedical research. This amorphous polymer is formed by a mesh of fibrils/lamellar structures with high porosity, is constituted by high molecular mass fractions, is highly sulfated and displays low viscosity, even in highly concentrated aqueous solutions. FTIR analysis confirmed the presence of several functional groups. We demonstrated that the ΔsigF polymer has strong biological activity, decreasing the viability of melanoma, thyroid and ovary carcinoma cells by inducing high levels of apoptosis, through p53 and caspase-3 activation. Therefore, the ΔsigF Synechocystis mutant is a promising platform for the sustainable production of biological active carbohydrate polymer(s) with the desired characteristics for biomedical applications.

The secretion signal peptide of the cyanobacterial extracellular protein HesF is located at its C-terminus.

Cyanobacteria are photosynthetic prokaryotes, capable of sustaining their growth by converting sunlight into chemical energy by fixing CO2 into organic matter. The cyanobacterium Anabaena sp. PCC 7120 is also capable of fixing atmospheric nitrogen, a metabolic process that occurs in specialized cells, the heterocysts. During the process of heterocyst differentiation, drastic morphological changes occur to prepare the future differentiated cell to accommodate the nitrogen fixation metabolism, which is a highly O2-sensitive process. Recently, we identified an unknown extracellular protein (termed HesF) in Anabaena sp. PCC 7120 and found it to be required for the proper deposition of the polysaccharide layers in the heterocyst cell wall. HesF is a non-classical type I secretion system (T1SS)-dependent secreted substrate, and its secretion signal remained elusive. Here, we report that the secretion signal of HesF is located in its C-terminus. We present evidence that a heterologous reporter protein fused with HesF's secretion signal could be successfully expressed in heterocysts and secreted to the extracellular medium, following hesF's native regulation. This represents the first time that the secretion signal of a cyanobacterial T1SS-dependent substrate is identified, and demonstrates the feasibility of using cyanobacteria for selected protein expression and secretion.

Description of new genera and species of marine cyanobacteria from the Portuguese Atlantic coast.

Aiming at increasing the knowledge on marine cyanobacteria from temperate regions, we previously isolated and characterized 60 strains from the Portuguese foreshore and evaluate their potential to produce secondary metabolites. About 15% of the obtained 16S rRNA gene sequences showed less than 97% similarity to sequences in the databases revealing novel biodiversity. Herein, seven of these strains were extensively characterized and their classification was re-evaluated. The present study led to the proposal of five new taxa, three genera (Geminobacterium, Lusitaniella, and Calenema) and two species (Hyella patelloides and Jaaginema litorale). Geminobacterium atlanticum LEGE 07459 is a chroococcalean that shares morphological characteristics with other unicellular cyanobacterial genera but has a distinct phylogenetic position and particular ultrastructural features. The description of the Pleurocapsales Hyella patelloides LEGE 07179 includes novel molecular data for members of this genus. The filamentous isolates of Lusitaniella coriacea - LEGE 07167, 07157 and 06111 - constitute a very distinct lineage, and seem to be ubiquitous on the Portuguese coast. Jaaginema litorale LEGE 07176 has distinct characteristics compared to their marine counterparts, and our analysis indicates that this genus is polyphyletic. The Synechococcales Calenema singularis possess wider trichomes than Leptolyngbya, and its phylogenetic position reinforces the establishment of this new genus.

Culture-dependent characterization of cyanobacterial diversity in the intertidal zones of the Portuguese coast: a polyphasic study.

Cyanobacteria are important primary producers, and many are able to fix atmospheric nitrogen playing a key role in the marine environment. However, not much is known about the diversity of cyanobacteria in Portuguese marine waters. This paper describes the diversity of 60 strains isolated from benthic habitats in 9 sites (intertidal zones) on the Portuguese South and West coasts. The strains were characterized by a morphological study (light and electron microscopy) and by a molecular characterization (partial 16S rRNA, nifH, nifK, mcyA, mcyE/ndaF, sxtI genes). The morphological analyses revealed 35 morphotypes (15 genera and 16 species) belonging to 4 cyanobacterial Orders/Subsections. The dominant groups among the isolates were the Oscillatoriales. There is a broad congruence between morphological and molecular assignments. The 16S rRNA gene sequences of 9 strains have less than 97% similarity compared to the sequences in the databases, revealing novel cyanobacterial diversity. Phylogenetic analysis, based on partial 16S rRNA gene sequences showed at least 12 clusters. One-third of the isolates are potential N(2)-fixers, as they exhibit heterocysts or the presence of nif genes was demonstrated by PCR. Additionally, no conventional freshwater toxins genes were detected by PCR screening.