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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.

Exploiting light to drive redox reactions is currently a hot topic since light is considered as an environmentally friendly source of energy. Consequently, cyanobacteria, which can use light e.g., for generating NADPH, are in the focus of research. Previously, it has been shown that various heterologous redox enzymes could be expressed in these microorganisms. Here we demonstrated the successful inducer-free expression of α-keto-acid dehydrogenases (L-HicDH and D-HicDH) from Lactobacillus confusus DSM 20196 and Lactobacillus paracasei DSM 20008 in Synechocystis sp. PCC 6803 ΔhoxYH mutant using replicative plasmids. While the L-HicDH showed poor activity limited by the amount of expressed enzyme, the D-HicDH was applied both in vivo and in vitro, transforming the selected α-keto acids to the corresponding optically pure (R)-α-hydroxy acids (ee >99%) in up to 53% and 90% conversion, respectively. Europäische Kommission 764920 Fundação para a Ciência e a Tecnologia (FCT) (DE-588b)6030623-3 1087594006 IF/00256/2015 Agency for Science Technology and Research (Singapore) (1087594006) SFRH/BD/117508/2016 Version of record

A functional, semi-synthetic and heterologously expressed [FeFe]-hydrogenase is generated in the photosynthetic microorganism Synechocystis, resulting in cells with sustained hydrogen production., [FeFe]-Hydrogenases are hydrogen producing metalloenzymes with excellent catalytic capacities, highly relevant in the context of a future hydrogen economy. Here we demonstrate the synthetic activation of a heterologously expressed [FeFe]-hydrogenase in living cells of Synechocystis PCC 6803, a photoautotrophic microbial chassis with high potential for biotechnological energy applications. H2-Evolution assays clearly show that the non-native, semi-synthetic enzyme links to the native metabolism in living cells.

Additional file 1: Fig. S1. Integration vector backbone sequence and plasmid map. Fig. S2. sYFP2 reporter construct sequence and plasmid map. Fig. S3. Colony PCR verification of the generated Synechocystis strains. Fig. S4. Negative fluorescent control (Synechocystis WT) for Fig. 2. Fig. S5. Agarose gel of the cell lysates used as template for RT-qPCR. Fig. S6. Colony PCR verification of genetic stability after six-week cultivation. Table S1. Conditional expression FCs (mRNA) of the selected target gene candidates. Table S2. Conditional expression FCs (mRNA) of additional gene targets. Table S3. List and descriptions of the PCR primers used in the study. Table S4. Integration constructs generated for sYFP2 expression. Table S5. Data of the RT-qPCR syfp2 analysis. Table S6. Amplicon-specific parameters in RT-qPCR.