Disrupted H2 synthesis combined with methyl viologen treatment inhibits photosynthetic electron flow to synergistically enhance glycogen accumulation in the cyanobacterium Synechocystis sp. PCC 6803.

Under nitrogen deprivation (-N), cyanobacterium Synechocystis sp. PCC 6803 exhibits growth arrest, reduced protein content, and remarkably increased glycogen accumulation. However, producing glycogen under this condition requires a two-step process with cell transfer from normal to -N medium. Metabolic engineering and chemical treatment for rapid glycogen accumulation can bypass the need for two-step cultivation. For example, recent studies indicate that individually disrupting hydrogen (H2) or poly(3-hydroxybutyrate) (PHB) synthesis, or treatment with methyl viologen (MV), effectively increases glycogen accumulation in Synechocystis. Here we explore the effects of disrupted H2 or poly(3-hydroxybutyrate) synthesis, together with MV treatment to on enhanced glycogen accumulation in Synechocystis grown in normal medium. Wild-type cells without MV treatment exhibited low glycogen content of less than 6% w/w dry weight (DW). Compared with wild type, disrupting PHB synthesis combined with MV treatment did not increase glycogen content. Disrupted H2 production without MV treatment yielded up to 11% w/w DW glycogen content. Interestingly, when combined, disrupted H2 production with MV treatment synergistically enhanced glycogen accumulation to 51% and 59% w/w DW within 3 and 7 days, respectively. Metabolomic analysis suggests that MV treatment mediated the conversion of proteins into glycogen. Metabolomic and transcriptional-expression analysis suggests that disrupted H2 synthesis under MV treatment positively influenced glycogen synthesis. Disrupted H2 synthesis under MV treatment significantly increased NADPH levels. This increased NADPH content potentially contributed to the observed enhancements in antioxidant activity against MV-induced oxidants, O2 evolution, and metabolite substrates levels for glycogen synthesis in normal medium, ultimately leading to enhanced glycogen accumulation in Synechocystis. KEY MESSAGE: Combining disrupted hydrogen-gas synthesis and the treatment by photosynthesis electron-transport inhibitor significantly enhance glycogen production in cyanobacteria.

Erythromycin mediates co-flocculation between cyanobacterium Synechocystis sp. PCC 6803 and filamentous fungi in liquid cultivation without organic compounds.

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 protected Synechocystis against the development of chlorosis. Transcriptomic analysis 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 CuSO4 stress can also mediate Synechocystis-fungi flocculation but at a lower flocculation efficiency than that caused by EM. The EM treatment may be applied in the co-culture between other cyanobacteria and fungi to mediate cell bio-flocculation.

Chemical Triggering Cyanobacterial Glycogen Accumulation: Methyl Viologen Treatment Increases Synechocystis sp. PCC 6803 Glycogen Storage by Enhancing Levels of Gene Transcript and Substrates in Glycogen Synthesis.

Two-stage cultivation is effective for glycogen production by cyanobacteria. Cells were first grown under adequate nitrate supply (BG11) to increase biomass and subsequently transferred to nitrogen deprivation (-N) to stimulate glycogen accumulation. However, the two-stage method is time-consuming and requires extensive energy. Thus, one-stage cultivation that enables both cell growth and glycogen accumulation is advantageous. Such one-stage method could be achieved using a chemical triggering glycogen storage. However, there is a limited study on such chemicals. Here, nine compounds previously reported to affect cyanobacterial cellular functions were examined in Synechocystis sp. PCC 6803. 2-Phenylethanol, phenoxyethanol, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and methyl viologen can stimulate glycogen accumulation. The oxidative stress agent, methyl viologen significantly increased glycogen levels up to 57% and 69% [w/w dry weight (DW)] under BG11 and -N cultivation, respectively. One-stage cultivation where methyl viologen was directly added to the pre-grown culture enhanced glycogen storage to 53% (w/w DW), compared to the 10% (w/w DW) glycogen level of the control cells without methyl viologen. Methyl viologen treatment reduced the contents of total proteins (including phycobiliproteins) but caused increased transcript levels of glycogen synthetic genes and elevated levels of metabolite substrates for glycogen synthesis. Metabolomic results suggested that upon methyl viologen treatment, proteins degraded to amino acids, some of which could be used as a carbon source for glycogen synthesis. Results of oxygen evolution and metabolomic analysis suggested that photosynthesis and carbon fixation were not completely inhibited upon methyl viologen treatment, and these two processes may partially generate upstream metabolites required for glycogen synthesis.