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:Photoautotrophic cyanobacteria assimilate the greenhouse gas carbon dioxide as their sole carbon source for producing useful bioproducts. However, harvesting the cells from their liquid media is a major bottleneck in the process. Thus, an easy-to-harvest method, such as auto-flocculation, is desirable. Here, we found that cyanobacterium Synechocystis sp. PCC 6803 co-flocculated with a natural fungal contamination in the presence of the antibiotic erythromycin (EM) but not without EM. The fungi in the co-flocculated biomass were isolated and found to consist of five species with the filamentous Purpureocillium lilacinum and Aspergillus protuberus making up 71% of the overall fungal population. The optimal co-cultivation for flocculation was an initial 5 mg (fresh weight) of fungi, an initial cell density of Synechocystis of 0.2 OD730, 10 µM EM, and 14 days of cultivation in 100 mL of BG11 medium with no organic compound. This yielded 248 ± 28 mg/L of the Synechocystis-fungi flocculated biomass from 560 ± 35 mg/L of total biomass, a 44 ± 2 % biomass flocculation efficiency. Furthermore, the EM treated Synechocystis cells in the Synechocystis-fungi flocculate had a normal cell color and morphology, while those in the axenic suspension exhibited strong chlorosis. Thus, the occurrence of the Synechocystis-fungi flocculation was mediated by EM, and the co-flocculation with the fungi helped Synechocystis to alleviate the negative effect of EM. Transcriptomic analysis of suspended and flocculated (with the fungi) Synechocystis cells suggested that the EM-mediated co-flocculation was a result of down-regulation of the minor pilin genes and up-regulation of several genes including the chaperone gene for pilin regulation, the S-layer protein genes, the exopolysaccharide-polymerization gene, and the genes for signaling proteins involved in cell attachment and abiotic-stress responses. The EM treatment may be applied in the co-culture between other cyanobacteria and fungi to mediate cell bio-flocculation.

Dataset Information

Isolation of intact and active FoF1 ATP synthase using a FLAG-tagged subunit from the cyanobacterium Synechocystis sp. PCC 6803

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Isolation of intact and active FoF1 ATP synthase using a FLAG-tagged subunit from the cyanobacterium <i>Synechocystis</i> sp. PCC 6803.

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