Abstract
BackgroundCyanobacteria 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.ResultsWe 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.ConclusionsA 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.
Highlights
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
Green-light induction of cell lysis The T4 phage-derived lysis genes encoding holin, endolysin, and antiholin [14,15,16,17] were introduced into Synechocystis on the broad host range vector pKT230 [18], which has been utilized for the recombinant expression in both freshwater and marine cyanobacterial strains [19,20,21,22,23,24]
To link the lysis genes to the endogenous CcaS/CcaR two-component system for green-light regulation, the region upstream of the Synechocystis cpcG2 gene was amplified by polymerase chain reaction (PCR) and inserted upstream of the T4 holin and endolysin genes (Figure 1)
Summary
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. 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. 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, which does not compete with terrestrial agricultural crops extraction with solvents. Non-mechanical methods, such as freeze-thaw, organic solvents, detergents, osmotic shock, acid, base, and enzyme reactions have been employed. Energy consumption is a concern for mechanical methods, while non-mechanical methods require additional steps for removing the chemicals, resulting in extra costs. More efficient and cost-effective methods must be designed
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