Glycogen Production in Marine Cyanobacterial Strain Synechococcus sp. NKBG 15041c.

An important feature offered by marine cyanobacterial strains over freshwater strains is the capacity to grow in seawater, replacing the need for often-limited freshwater. However, there are only limited numbers of marine cyanobacteria that are available for genetic manipulation and bioprocess applications. The marine unicellular cyanobacteria Synechococcus sp. strain NKBG 15041c (NKBG15041c) has been extensively studied. Recombinant DNA technologies are available for this strain, and its genomic information has been elucidated. However, an investigation of carbohydrate production, such as glycogen production, would provide information for inevitable biofuel-related compound production, but it has not been conducted. In this study, glycogen production by marine cyanobacterium NKBG15041c was investigated under different cultivation conditions. NKBG15041c yielded up to 399 µg/ml/OD730 when cells were cultivated for 168 h in nitrogen-depleted medium (marine BG11ΔN) after medium replacement (336 h after inoculation). Cultivation under nitrogen-limited conditions also yielded an accumulation of glycogen in NKBG15041c cells (1 mM NaNO3, 301 µg/ml/OD730; 3 mM NaNO3, 393 µg/ml/OD730; and 5 mM NaNO3, 328 µg/ml/OD730) under ambient conditions. Transcriptional analyses were carried out for 13 putative genes responsible for glycogen synthesis and catabolism that were predicted based on homology analyses with Synechocystis sp. PCC 6803 (PCC6803) and Synechococcus sp. PCC7002 (PCC7002). The transcriptional analyses revealed that glycogen production in NKBG15041c under nitrogen-depleted conditions can be explained by the contribution of both increased carbon flux towards glycogen synthesis, similar to PCC6803 and PCC7002, and increased transcriptional levels of genes responsible for glycogen synthesis, which is different from the conventionally reported phenomenon, resulting in a relatively high amount of glycogen under ambient conditions compared to PCC6803 and PCC7002.

Construction of a Miniaturized Chromatic Acclimation Sensor from Cyanobacteria with Reversed Response to a Light Signal.

Cyanobacteria harbor unique photoreceptors, designated as cyanobacteriochromes (CBCRs). In this study, we attempted to engineer the chromatic acclimation sensor CcaS, a CBCR derived from the cyanobacterium Synechocystis sp. PCC 6803. The wild-type CcaS induces gene expression under green light illumination and represses it under red light illumination. We focused on the domain structure of CcaS, which consists of an N-terminal transmembrane helix; a GAF domain, which serves as the sensor domain; a linker region (L1); two PAS domains; a second linker region (L2); and a C-terminal histidine kinase (HK) domain. Truncated versions of the photoreceptor were constructed by removing the L1 linker region and the two PAS domains, and fusing the GAF and HK domains with a truncated linker region. Thus constructed "miniaturized CcaSs" were grouped into four distinct categories according to their responses toward green and red light illumination, with some showing improved gene regulation compared to the wild type. Remarkably, one of the miniaturized CcaSs induced gene expression under red light and repressed it under green light, a reversed response to the light signal compared to wild type CcaS. These characteristics of engineered photoreceptors were discussed by analyzing the CcaS structural model.

Anionic metabolite biosynthesis enhanced by potassium under dark, anaerobic conditions in cyanobacteria.

Potassium (K(+)) is an essential macronutrient for all living organisms including cyanobacteria. Cyanobacteria are a group of bacteria performing oxygenic photosynthesis, widely studied in basic and applied sciences. The primary metabolism of the unicellular cyanobacterium Synechocystis sp. PCC 6803 is altered by environmental conditions, and it excretes organic acids and hydrogen under dark, anaerobic conditions. Here we demonstrated that K(+) widely changes the primary carbon metabolism of this cyanobacterium. Succinate and lactate excretion from the cells incubated under dark, anaerobic conditions was enhanced in the presence of K(+), while hydrogen production was repressed. The addition of K(+) and the genetic manipulation of acetate kinase AckA and an RNA polymerase sigma factor SigE additively increased succinate and lactate production to 141.0 and 217.6 mg/L, which are 11 and 46 times, compared to the wild-type strain without K(+), respectively. Intracellular levels of 2-oxoglutarate, succinate, fumarate, and malate increased by K(+) under dark, anaerobic conditions. This study provides the evidence of the considerable effect of K(+) on the biosynthesis of anionic metabolites in a unicellular cyanobacterium.

Development of a light-regulated cell-recovery system for non-photosynthetic bacteria.

BACKGROUND: Recent advances in the understanding of photosensing in biological systems have enabled the use of photoreceptors as novel genetic tools. Exploiting various photoreceptors that cyanobacteria possess, a green light-inducible gene expression system was previously developed for the regulation of gene expression in cyanobacteria. However, the applications of cyanobacterial photoreceptors are not limited to these bacteria but are also available for non-photosynthetic microorganisms by the coexpression of a cyanobacterial chromophore with a cyanobacteria-derived photosensing system. An Escherichia coli-derived self-aggregation system based on Antigen 43 (Ag43) has been shown to induce cell self-aggregation of various bacteria by exogenous introduction of the Ag43 gene. RESULTS: An E. coli transformant harboring a plasmid encoding the Ag43 structural gene under a green light-regulated gene expression system derived from the cyanobacterium Synechocystis sp. PCC6803 was constructed. Ag43 was inserted downstream of the cpcG 2 promoter P cpcG2 , and its expression was regulated by green light induction, which was achieved by the functional expression of cyanobacterial CcaS/CcaR by coexpressing its chromophore synthesis gene cassette in E. coli. E. coli transformants harboring this designed system self-aggregated under green light exposure and precipitated, whereas transformants lacking the green light induction system did not. The green light induction system effectively functioned before the cell culture entered the stationary growth phase, and approximately 80 % of the cell culture was recovered by simple decantation. CONCLUSION: This study demonstrated the construction of a cell recovery system for non-photosynthetic microorganisms induced by exposure of cells to green light. The system was regulated by a two-component regulatory system from cyanobacteria, and cell precipitation was mediated by an autotransporter protein, Ag43. Although further strict control and an increase of cell recovery efficiency are necessary, the system represents a novel tool for future bioprocessing with reduced energy and labor required for cell recovery.

A green-light inducible lytic system for cyanobacterial cells.

BACKGROUND: Cyanobacteria are an attractive candidate for the production of biofuel because of their ability to capture carbon dioxide by photosynthesis and grow on non-arable land. However, because huge quantities of water are required for cultivation, strict water management is one of the greatest issues in algae- and cyanobacteria-based biofuel production. In this study, we aim to construct a lytic cyanobacterium that can be regulated by a physical signal (green-light illumination) for future use in the recovery of biofuel related compounds. RESULTS: We introduced T4 bacteriophage-derived lysis genes encoding holin and endolysin under the control of the green-light regulated cpcG2 promoter in Synechocystis sp. PCC 6803. When cells harboring the lysis genes were illuminated with both red and green light, we observed a considerable decrease in growth rate, a significant increase in cellular phycocyanin released in the medium, and a considerable fraction of dead cells. These effects were not observed when these cells were illuminated with only red light, or when cells not containing the lysis genes were grown under either red light or red and green light. These results indicate that our constructed green-light inducible lytic system was clearly induced by green-light illumination, resulting in lytic cells that released intracellular phycocyanin into the culture supernatant. This property suggests a future possibility to construct photosynthetic genetically modified organisms that are unable to survive under sunlight exposure. Expression of the self-lysis system with green-light illumination was also found to greatly increase the fragility of the cell membrane, as determined by subjecting the induced cells to detergent, osmotic-shock, and freeze-thaw treatments. CONCLUSIONS: A green-light inducible lytic system was constructed in Synechocystis sp. PCC 6803. The engineered lytic cyanobacterial cells should be beneficial for the recovery of biofuels and related compounds from cells with minimal effort and energy, due to the fragile nature of the induced cells. Furthermore, the use of light-sensing two-component systems to regulate the expression of exogenous genes in cyanobacteria promises to replace conventional chemical inducers in many bioprocess applications, impacting the limiting water management issues.

Screening of Aspergillus-derived FAD-glucose dehydrogenases from fungal genome database.

Aspergillus-derived FAD-dependent glucose dehydrogenases (FADGDHs) were screened from fungal genomic databases, primarily by searching for putative homologues of the Aspergillus niger-derived glucose oxidase (GOD). Focusing on a GOD active-site motif, putative proteins annotated as belonging to the glucose methanol choline (GMC) oxidoreductase family were selected. Phylogenetic analysis of these putative proteins produced a GOD clade, which includes the A. niger and Penicillium amagasakiens GODs, and a second clade made up of putative proteins showing 30-40% homology with GOD. The genes encoding the proteins from the second clade were functionally expressed in Escherichia coli, resulting in dye-mediated glucose dehydrogenase (GDH) activity but not GOD activity. These results suggest that the putative proteins belonging to the second clade are FADGDHs. The 3D structure models of these FADGDHs were compared with the 3D structure of GOD.