Project description:Cyanobacteria are phototrophic prokaryotes that can convert inorganic carbon as CO2 into organic carbon compounds at the expense of light energy. In addition, they need only a few inorganic nutrients and can be cultivated in high densities using non-arable land and seawater. This features qualified cyanobacteria as attractive organisms for the production of third generation biofuels as part of the development of future CO2-neutral energy production. Synechocystis sp. PCC 6803 represents one of the most widely used cyanobacterial model strains. On the basis of its available genome sequence and genetic tools, many strains of Synechocystis have been generated that produce different biotechnological products. Efficient isoprene production is an attractive goal, since this compound represents not only an energy-rich biofuel but is also used as chemical feedstock. Here, we report on our attempts to generate isoprene-producing strains of Synechocystis. The cDNA of a codon-optimized plant isoprene synthase (IspS) was cloned under the control of different Synechocystis promoters, which ensure strong constitutive or light-regulated ispS expression. The expression of the ispS gene was quantified by qPCR, whereas the amount of isoprene was quantified using GC-MS. Incubation of our strains at different salt conditions had marked impact on the isoprene production rates. Under low salt conditions, a good correlation was found between ispS expression and isoprene production rate. However, the cultivation of isoprene production strains under salt-supplemented conditions decreased isoprene production despite the fact that ispS expression was salt-stimulated. The characterization of the metabolome of isoprene producing strains indicated that isoprene production might be limited by insufficient precursor levels. Our isoprene production rates under low salt conditions were 2 - 6.5times higher compared to the previous report of Lindberg et al. (2010). These results can be used to guide future attempts establishing the isoprene production with cyanobacterial host systems.
Project description:Ethylene is a gaseous signal sensed by plants and bacteria. Heterologous expression of the ethylene-forming enzyme (EFE) from Pseudomonas syringae in cyanobacteria leads to the production of ethylene under photoautotrophic conditions. The recent characterization of an ethylene responsive signaling pathway affecting phototaxis in the cyanobacterium Synechocystis sp. PCC 6803 implies that biotechnologically relevant ethylene synthesis may induce regulatory processes which are not related to changes in the metabolism. Here we provide data that endogenously produced ethylene accelerates movement of cells towards light. Microarray analysis demonstrates that ethylene deactivates transcription from the csiR1/lsiR promoter which is under control of the two-component system consisting of the ethylene and UV-A-sensing histidine kinase UirS and the DNA-binding response regulator UirR. Surprisingly, only very few other transcriptional changes were detected in the microarray analysis providing no direct hints to possible bottlenecks in phototrophic ethylene production.
Project description:In cyanobacteria DNA supercoiling varies over the diurnal light/dark cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knockdown of gyrase subunits gyrA, gyrB and overexpression of topoisomerase I (TopoI) topA and analyzed the transcriptional response to gyrase knock-downs (endpoint in triplicate) and topoisomerase I overexpression (endpoint in triplicate, and 19 time points time series before and after induction) in Synechocystis sp. PCC 6803 via RNA-seq of coding RNA. In detail, Illumina Ribo-Zero Plus rRNA Depletion Kit was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was evaluated with the RNA Pico 6000 kit on the Agilent 2100 Bioanalyzer. RNA was free of detectable rRNA. Preparation of cDNA libraries was performed according to the manufacturer’s instructions for the TruSeq stranded mRNA kit (Illumina, San Diego, CA, United States). Subsequently, each cDNA library was sequenced on an Illumina NextSeq 500 system (2 x 75 nt PE high output v2.5).
Project description:In vivo recruitment analysis and a mutant strain without any group 2 σ factors reveal roles of different σ factors in cyanobacteria
Project description:The protein abundance of Synechocystis in response to the given copper-iron combination was examined, then followed by comparative proteomic analyses to reveal the proteins associated with copper and iron stress. These data would provide a theoretical basis for understanding the relationship between copper and iron in cyanobacteria at the protein level and shed light on the role of these two metal elements in energy metabolism and biomass accumulation of cyanobacteria.